Thermotolerant phytase for animal feed

ABSTRACT

The invention provides a synthetic phytase polynucleotide which is optimized for expression in plants and which encodes at thermotolerant phytase, as well as isolated thermotolerant phytase enzyme. Also provided are feed or food products comprising a thermotolerant phytase, and transgenic plants which express the thermotolerant phytase. Further provided are methods for making and using thermotolerant phytases, e.g., a method of using a thermotolerant phytase in feed and food processing.

RELATED APPLICATION

This application is a division of U.S. Ser. No. 11/412,185, filed Apr.26, 2006, which is a division of U.S. Ser. No. 10/334,671, filed Dec.30, 2002, which claims priority to Application No. 60/344,476, filedDec. 28, 2001, all of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention generally relates to the field of molecularbiology, and more specifically, to methods of making and using athermotolerant phytase.

BACKGROUND OF THE INVENTION

Phytases (myo-inositol hexakisphosphate phosphohydrolase: EC 3.1.3.8)are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) tomyo-inositol and inorganic phosphate. The enzymes are known to bevaluable feed additives. At the close of the twentieth century, annualsales of phytase as an animal feed additive were estimated to be $100million and growing.

Poultry and pig diets are currently based primarily on cereals, legumes,and oilseed products. About two-thirds of phosphorus (P) present inthese feedstuffs occur as phytates, the salts of phytic acid(myo-inositol hexakisphosphate, InsP6) (Jongbloed et al., 1993). Phytatephosphorus in plants is a mixed calcium-magnesium-potassium salt ofphytic acid that is present as chelate and its solubility is very low(Pallauf and Rimbach, 1997). Phosphorus in this form is poorlydigestible/available for monogastric animals such as human, swine, andpoultry.

For the utilization of phytate phosphorus and minerals and traceelements bound in phytic acid complexes, hydrolysis of the ester-typebonded phosphate groups of phytic acid by phytase is necessary (Rimbachet al., 1994). Phytases belong to a special group of phosphatases whichare capable of hydrolyzing phytate to a series of lower phosphate estersof myo-inositol and phosphate. Two types of phytases are known:3-phytase and 6-phytase, indicating the initial attack of thesusceptible phosphate ester bond. Although monogastric animals lacksufficient phytase to effectively utilize phytate phosphorous, manyfungi, bacteria and yeasts produce phytase that can be used tosupplement animal rations.

The beneficial effects of supplementary phytases on phosphorusdigestibility and animal performance have been well documented (Mroz etal., 1994; Kornegay et al., 1996; Rao et al., 1999; Ravindran et al.,1999). However, most of these studies have been performed on an ad hocbasis with often only superficial information of the enzymes provided asmarketing strategies by the manufacturers. The efficacy of any enzymepreparation depends not only on the type, inclusion rate and level ofactivity present, but also on the ability of the enzyme to maintain itsactivity in the different conditions encountered through thegastrointestinal tract and the conditions used for the pre-treatment ofa food or feed formulation.

Although numerous phytases are available for use as supplements, many ofthe enzymes have certain disadvantages. For example, many of thecurrently used phytases lose activity during feed pelleting process dueto heat treatment. Additionally, many of the currently used phytases arenot adequate in diets containing low levels of supplemental calciumphosphate. In addition, in many instances, there is a high cost ofproduction associated with the microbially expressed enzymes.

Thus, what is needed is a phytase with improved properties for animalfeed and food processing as well as an economical procedure forproducing the phytase. One method of producing a more economical phytasewould be to use recombinant DNA techniques to produce transgenic plantsor plant organs capable of expressing phytase which could then be addedas such to animal feed or human food for direct consumption.Alternatively, the phytase could be extracted and, if desired, purifiedfor the desired application.

SUMMARY OF THE INVENTION

Accordingly, the invention provides methods of preparing and using anucleic acid molecule (polynucleotide) which encodes a thermotolerantphytase, i.e., a thermotolerant phytase which retains at least 40%activity after 30 minutes at about 60° C., and which has a high specificactivity, i.e., at least about 200 U/mg at 37° C. and at acid pH, e.g.,pH 4.5.

In one embodiment, the invention provides a method to prepare athermotolerant phytase. The method comprises expressing in a plant hostcell an expression cassette comprising a promoter operably linked to anucleic acid molecule encoding a thermotolerant phytase which retains atleast 40% activity after 30 minutes at 60° C. and has a specificactivity of greater than 200 U/mg at pH 4.5 and 37° C. In a preferredembodiment, the method further comprises isolating the thermotolerantphytase. The plant host cell may be monocotyledonous, such as a maize orwheat cell or dicotyledenous, such as a soybean cell.

In a preferred embodiment, the plant cell which is employed to preparethe recombinant thermotolerant phytase yields a glycosylated form of therecombinant thermotolerant phytase.

The invention also provides a polynucleotide sequence encoding thethermotolerant phytase wherein the polynucleotide sequence is optimizedfor expression in a plant cell, namely a maize cell.

It is preferred that the polynucleotide that encodes the thermotolerantphytase (the first polynucleotide) is operably linked to at least oneregulatory sequence, such as a promoter, an enhancer, an intron, atermination sequence, or any combination thereof, and, optionally, to asecond polynucleotide encoding a signal sequence, which directs theenzyme encoded by the first polynucleotide to a particular cellularlocation e.g., an extracellular location. Promoters can be constitutivepromoters, such as a ubiquitin promoter or inducible (conditional)promoters. Promoters may be tissue-specific. In a preferred embodiment,the promoter is an endosperm-specific promoter such as a maize γ-zeinglutelin-2 promoter, and preferably the maize 27-KD γ-zein glutelin-2promoter, or a maize ADP-glucose pyrophosphorylase promoter. Thepromoter may be an embryo-specific promoter such as a maize globulin 1or maize oleosin 18 KD promoter. Exemplary promoters include, but arenot limited to, SEQ ID NO:8 and SEQ ID NO:9.

Preferably, the thermotolerant phytase of the invention has at least 40%activity at about 60° C. for 30 minutes, more preferably at least 40%activity at about 65° C. for 30 minutes, even more preferably at least35% activity at 70° C. for 30 minutes, and which has a specific activityof at least 400 U/mg, more preferably at least 600 U/mg, and even morepreferably at least 800 U/mg, at 37° C. and at acid pH, e.g., less thanpH 5.0 and more preferably less than pH 4.0 and greater than pH 1.5. Anexemplary thermotolerant phytase of the invention is provided in SEQ IDNO: 1.

Also provided by the invention are vectors which comprise the expressioncassette or polynucleotide of the invention and plant cells andtransformed plant cells comprising the polynucleotide, expressioncassette or vector of the invention. A vector of the invention canencode more than one polypeptide including more than one thermotolerantphytase or may encode a fusion polypeptide comprising the thermotolerantphytase of the invention, and a plant cell may comprise one or morevectors of the invention. The transformed plant cells of the inventionare useful for preparing the recombinant thermotolerant phytase of theinvention. Accordingly, the invention provides thermotolerant phytaseisolated from the transformed plant cells of the invention, as well assynthetically prepared enzyme.

A fusion polypeptide prepared by the methods of the invention preferablycomprises a signal sequence. The signal sequence, operably linked to thephytase gene, targets the phytase to an amyloplast, endoplasmicreticulum, apoplast, or starch granule of the cell. Exemplary signalsequences include, but are not limited to, the N-terminal sequence fromwaxy, the N-terminal sequence from γ-zein, a starch binding domain suchas a waxy starch encapsulating domain. In a preferred embodiment, thefusion polypeptide prepared and employed in the methods of the inventioncomprises SEKDEL signal sequence operably linked to the C-terminus ofthe thermotolerant phytase.

The thermotolerant phytase provided by the invention has a half-life ofgreater than 25 minutes at a pH greater than 2.0 and less than 4.0.

Further provided by the invention are methods for formulation ofthermotolerant phytases, phytase formulations or formulated enzymemixtures. The recombinant thermotolerant phytase or formulations thereofmay be added as a supplement to food or animal feed or to components offood and feed prior to, during, or after food or feed processing.Preferably, the recombinant thermotolerant phytase of the invention isadded to a mixture of feed components prior to and/or during heat (e.g.,steam) conditioning in a pellet mill. Thus, the invention includesmethods of making and using a thermotolerant phytase.

Further, as a phytase of the invention is capable of surviving the heatconditioning step encountered in a commercial pellet mill during feedformulation, the invention provides a method of preparing animal feed,e.g., hard granular feed pellets comprising the thermotolerant phytase.To make feed, the formulated phytase may be mixed with feed components,the mixture steam conditioned in a pellet mill such that at least 50% ofthe pre-heat treated enzymatic activity is retained, and the feedextruded through a pellet dye. The phytase may thus be used as asupplement in animal feed by itself, in addition with vitamins,minerals, other feed enzymes, agricultural co-products (e.g., wheatmiddlings or corn gluten meal), or in a combination therewith. Theenzyme may also be added to mash diets, i.e., diets that have not beenthrough a pelletizer.

Because the currently available commercial phytase enzymes are notthermotolerant, they are often applied post pelleting, generally viaspraying an enzyme solution onto pelleted feed. Some of the problemsassociated with spraying methods are that only a low percentage of thepellets are contacted with enzyme, the enzyme is only present on thesurface of the coated pellets, and feed mills need to invest in andoperate complex spraying machinery. In contrast, the thermotolerantphytase of the invention, which has about an 8-fold higher specificactivity than other commercially available enzymes, may be added priorto pelleting, thereby facilitating production of a feed with an improveddistribution of the enzyme. Moreover, feed comprising the thermotolerantphytase of the invention may have a longer shelf-life than feed sprayedwith phytase, as the spraying process introduces moisture which cansupport fungal and bacterial growth during storage. Further, the higherspecific activity of the thermotolerant phytase of the invention allowsfeed manufacturers to use significantly lower phosphate levels in feed.For example, it is currently recommended that diets supplemented withthe available commercial phytases use a basal level of 0.45% inorganicphosphate. The thermotolerant phytase of the invention may be used witha lower phosphate supplementation, e.g., about 0.225% in poultry diets.

The invention thus provides a method of preparing animal feed comprisingproviding a mixture comprising one or more feed components and apreparation comprising the thermotolerant phytase of the invention, andtreating the mixture under appropriate conditions of temperature andmoisture so as to hydrolyze phytic acid which is present in the mixture.Also provided is animal feed prepared by such a method.

Further provided is a method of preparing a thermotolerant phytasecontaining composition for feed formulation comprising combining aliquid solution comprising the thermotolerant phytase of the inventionand meal flour, e.g., soy meal flour, to yield a mixture; andlyophilizing the mixture to yield a lyophilized composition. In anadditional embodiment, the method further comprises combining thelyophilized composition with other feed components to yield a furthermixture. Lyophilized compositions prepared according to these methodsare also provided by the invention.

The invention further provides a method in which a mixture comprising ananimal feed component and a preparation comprising the thermotolerantphytase of the invention is treated with heat, preferably at atemperature greater than 50° C., so as to yield a heat-treated animalfeed mixture. Heat-treated animal feed prepared by the method is alsoprovided. The phytase preparation may be a liquid or a solidpreparation, and preferably comprises less than about 1% inorganicphosphate. Preferably, the phytase preparation is transgenic plantmaterial. The transgenic plant material is preferably corn grain,cracked corn, corn flour, or an enzyme extract prepared from corn.

In one embodiment, a liquid solution comprising the thermotolerantphytase of the invention is combined with soy meal flour to yield amixture and the mixture is then lyophilized. The mixture, whichpreferably comprises less than 0.45% inorganic phosphate, may alsocomprise at least one vitamin, mineral, an enzyme other than athermotolerant phytase, an organic acid, a probiotic product, anessential oil or a grain processing co-product. The heat-treated feedmay be further processed, for example, by extruding the heat-treatedfeed through a pellet mill to yield pelletized animal feed, which isalso encompassed by the invention.

Also provided is an animal feed composition comprising thethermotolerant phytase prepared by the methods of the invention. Inparticular, the invention provides an animal feed composition comprisingthe thermotolerant phytase, prepared according to the methods of theinvention, which phytase has a specific activity of greater than 400U/mg at pH 4.5 and 37° C., and preferably greater than 600 U/mg at pH4.5 and 37° C., and more preferably greater than 800 U/mg at pH 4.5 and37° C. In a preferred embodiment, the animal feed composition comprisesa thermotolerant phytase which has a half-life of greater than 25minutes at a pH greater than 2.0 and less than 4.0.

Also provided is an enzyme feed additive or a food additive comprising athermotolerant phytase prepared according to the methods of theinvention. In preferred embodiments, the feed and food additivescomprise a thermotolerant phytase which has a specific activity ofgreater than 400 U/mg at pH 4.5 and 37° C., and preferably greater than600 U/mg at pH 4.5 and 37° C., and more preferably greater than 800 U/mgat pH 4.5 and 37° C. In additional preferred embodiments, the animalfeed and food additives comprise a thermotolerant phytase which has ahalf life of greater than 25 minutes at a pH greater than 2.0 and lessthan 4.0.

Also provided is a method of reducing/decreasing the feed conversionratio and increasing the weight gain of an animal comprising feeding toan animal a feed comprising the thermotolerant phytase. In a preferredembodiment, the method comprises feeding to an animal a feed comprisinginorganic phosphate at below 0.45% and the thermostable phytase of theinvention in an amount effective to improve the feed conversion ratio orthe weight gain in the animal. In additional embodiments, it ispreferred that the thermostable phytase has a half-life of about 30minutes in the digestive tract of the animal.

Further provided is a method of minimizing dietary requirements ofphosphorus, e.g., inorganic phosphorous, in an animal. The methodcomprises feeding to an animal a feed comprising the thermotolerantphytase of the invention in an amount effective to increase thebioavailability of phosphorus, preferably the bioavailability ofinorganic phosphorous, in the feed to the animal. Also provided is amethod of enhancing the utilization of phosphorus present in feed for ananimal, which method comprises feeding to the animal a feed comprisingthe thermotolerant phytase of the invention in an amount effective toincrease the bioavailability of phosphorus in the feed to the animal. Inadditional embodiments of these methods, the phytase has a half-life ofabout 30 minutes in the digestive tract of the animal.

In addition, the invention provides a method of decreasing the phosphatelevels in excreta from an animal comprising feeding to the animal a feedcomprising the thermotolerant phytase of the invention in an amounteffective to lower levels of phosphate in the excreta of the animal. Ina preferred embodiment, the invention provides a method of decreasingthe phosphate levels in excreta from an animal comprising feeding to theanimal a feed comprising less than 0.45% inorganic phosphorus and thethermotolerant phytase of the invention in an amount effective to lowerlevels of phosphate in the excreta of the animal. In additionalembodiments of these methods, the phytase has a half-life of about 30minutes in the digestive tract of the animal.

The invention also provides a method of improving the nutritive value ofanimal feed or human food. The method comprises adding thethermotolerant phytase of the invention during the preparation of animalfeed or human food. Also provided is a method of preparing human foodcomprising providing a mixture of a food component and a preparationcomprising the thermotolerant phytase of the invention; and treating themixture under appropriate conditions of temperature and moisture tofacilitate the hydrolysis of phytic acid present in the mixture. Treatedhuman food prepared according to this method is also encompassed by theinvention. The treated human food has a reduced phytic acid contentrelative to the phytic acid content in corresponding human food that isnot treated.

The invention also provides a method of improving the processing ofgrain comprising adding the thermotolerant phytase of the inventionduring processing of the grain. The method may be used to improve theprocessing of all grain, and is preferably used to improve theprocessing of corn, wheat, soybean, canola, or sugarcane.

The invention further provides a method of improving the nutritive valueof a processed grain product or a method of processing grain comprisingadding the thermotolerant phytase of the invention to the grain productduring grain processing in an amount effective to improve the nutritivevalued of the feed. In a preferred embodiment, the grain is corn and thegrain processing method is wet milling and the products of theprocessing are corn gluten feed, corn gluten, and corn starch. Inadditional preferred embodiments, the grain is corn, wheat, soybean,canola, or sugarcane. In other preferred embodiments, the grain is anoilseed, such as soybean or canola or oilseed rape, and the processedgrain product is the oilseed meal.

The invention further provides a method of preparing a thermotolerantphytase containing composition for food formulation comprising combininga liquid solution comprising the thermotolerant phytase of the inventionand meal flour to yield a mixture; and lyophilizing the mixture to yielda lyophilized composition. The invention also provides a lyophilizedcomposition prepared by such method. In a preferred embodiment, themethod further comprises combining the lyophilized composition withother food components to yield a further mixture. In one preferredembodiment, the thermotolerant phytase containing composition comprisesa thermotolerant phytase which has a specific activity of greater than800 U/mg at pH 4.5 and 37° C. In another preferred embodiment, thethermotolerant phytase has a half life of greater than 25 minutes at apH greater than 2.0 and less than 4.0

In additional aspects, the invention provides methods to prepare atransformed plant cell, plant part and plant, which express thethermotolerant phytase of the invention. The invention also encompassesthe transgenic plant cell, plant part and plant produced by thesemethods. In preferred embodiments, the method comprises introducing intoa plant cell an expression cassette comprising a promoter operablylinked to a nucleic acid molecule encoding the thermotolerant phytaseand obtaining a transgenic plant from the transformed plant cell. Thethermotolerant phytase preferably retains at least 40% activity after 30minutes at 60° C. and has a specific activity of greater than 200 U/mgat pH 4.5 and 37° C. More preferably, the thermotolerant phytase has aspecific activity of greater than 400 U/mg at pH 4.5 and 37° C. Stillmore preferably, the thermotolerant phytase has a specific activity ofgreater than 600 U/mg at pH 4.5 and 37° C. Even more preferably,thermotolerant phytase has a specific activity of greater than 800 U/mgat pH 4.5 and 37° C.

The transformed plant cell, plant part or plant may be a dicot cell or amonocot cell, preferably a cereal cell, and more preferably a maize orwheat cell, or a soybean cell.

In preferred embodiments, the method is employed to prepare transgenicplant cell, plant part, or plant comprising the thermotolerant phytaseset forth in SEQ ID NO:1. The transgenic plant cell, plant part, andplant produced therein is included within the scope of the invention.

In another preferred embodiment of the method of the invention, thethermotolerant phytase is expressed in the seed of the plant. The seedof such a plant is encompassed within the scope of the invention.

Also within the scope of this invention is a transformed plant cell,plant part, and plant comprising the expression cassette of theinvention. As described previously, the expression cassette comprises apromoter operably linked to a nucleic acid molecule encoding thethermotolerant phytase of the invention. The promoter may be an embryospecific promoter, such as a maize globulin-1 promoter or a maizeoleosin 18 KD promoter, and is preferably an endosperm-specificpromoter, such as a maize ADP-glucose phosphorylase promoter or a maizeγ-zein promoter.

The invention also encompasses a transformed plant cell, plant part anda plant comprising a nucleic acid molecule which encodes a fusionpolypeptide comprising the thermotolerant phytase of the invention. In apreferred embodiment, the plant comprises a fusion polypeptide comprisesa γ-zein N-terminal signal sequence operably linked to thethermotolerant phytase. In another preferred embodiment, the plantcomprises a fusion polypeptide comprising SEKDEL operably linked to theC-terminus of the thermotolerant phytase. In another preferredembodiment, the plant comprises a fusion polypeptide comprising anN-terminal waxy amyloplast targeting peptide operably linked to thethermotolerant phytase. In another preferred embodiment, the plantcomprises a fusion polypeptide comprising a waxy starch encapsulatingdomain operably linked to the C-terminus of the thermotolerant phytase.

The invention also encompasses a product of the plant of the invention,which product comprises the thermotolerant phytase. The product ispreferably a seed, grain or fruit. The product may be a plant, and inparticular a hybrid plant or an inbred plant. The product may also be agrain processing product comprising the thermotolerant phytase of theinvention, such as the corn grain processing product and the oilseedgrain processing product previously described herein or an oilseedprocessing product.

Animals within the scope of the invention include polygastric animals,e.g., calves, as well as monogastric animals such as swine, poultry(e.g., chickens, turkeys, geese, ducks, pheasant, grouse, quail andostrich), equine, ovine, caprine, canine and feline, as well as fish andcrustaceans. Preferred feed or animal feed prepared and/or employed inthe invention include poultry and swine feed.

The levels of phytase in feed or food are preferably about 50 to 5000U/kg, more preferably 100 to 1200 U/kg, or 300 to 1200 U/kg.

The invention also provides a transformed plant produced by the methodsof the invention. Preferably, the transformed plant is a corn, wheat orsoybean plant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates that expression of the codon optimized maize phytasegene (Nov9X) encodes a functional phytase that accumulates in maizeseed. Top: each bar represents the total phosphate extracted from sixkernels from plants segregating for the Nov9X transgene. Bottom: totalphytase activity (background phosphate was substracted beforecalculating activity). 1 unit=μmol phosphate liberated/min by totalextract of 6 kernels. 264A8C#14 is the negative control (lacking Nov9Xtransgene); 305A13 contains plasmid pNOV4057; 305B11 contains plasmidpNOV4061.

FIG. 2 demonstrates that recombinant Nov9X phytase activity produced inmaize is heat stable. Units are defined as in described in FIG. 1. A.Total phytase activity extracted. B. SDS-PAGE gel of extracts stainedwith Coomassie blue.

FIG. 3 provides a comparison of phytase levels in extracts of flour fromdried kernels. All plants contain either pNOV4057 or pNOV4061. Top:phosphate concentration of reactions. Bottom: Phytase units are reportedas μmoles phosphate liberated/mg protein/min. Nov9X copy # top 4:305A24A, 2copies; 305B11A, 20A, 27A all >3 copies.

FIG. 4 shows the accumulation of phytase (Nov9X) produced in maize. Thefigures provide a comparison of phytase activity in corn floursuspensions and flour extracts. 266B-2E is a negative control. Thephosphate concentration of all samples are shown in the top graph.Phytase units were calculated after correcting for the endogenousphosphate present in the negative control 266B-2E. Duplicates of eachcontrol and treatment are shown. The phosphate concentration of thenegative control was subtracted from that of the treatments in order todetermine phosphate released due to phytase activity (bottom graphs).

FIG. 5 demonstrates endosperm-specific expression of Nov9X. The figuresshows phytase (Nov9X) activity in extracts of T2 endosperm and embryo.Duplicate samples were extracted (sets 1 & 2).

FIG. 6 shows that maize-expressed Nov9X phytase is stable during heatingof endosperm extracts and is highly enriched in the soluble fraction ofthe heated supernatant. A. Phytase activity of unheated and heatedsamples. B. Phytase specific activity of unheated and heated samples.

FIG. 7 shows phytase activities (FTU/g) of several lines containing 2events. Each data point represents phytase activity extracted from 1 gflour obtained by pulverizing 10 kernels. Duplicates samples of 10kernels were pulverized for each line (samples 1 & 2). Inbred IDsubstitutes for pollinator, maintainer, and sterile as used in previousversion.

FIGS. 8A, 8B, and 8C show feed conversion ratios (FCRs) from three21-day chicken feeding trials demonstrate benefit of corn phytasesupplementation. FCRs are reported as LSmeans. Available phosphorus:positive controls, 0.400%; negative controls, 0.225%. Phytasesupplemented diets were prepared by adding milled transgenic corn tosamples of low-phosphate diets (0.225% available phosphorus=negativecontrol). The difference between the negative control diet and theenzyme-supplemented diets is the addition of milled transgenic corncontaining Nov9X phytase.

FIG. 8A: Phytase was formulated by grinding whole transgenic cornkernels to flour. The flour was then added directly to mash feed (lowphosphate) and mixed thoroughly. Animals were then fed mash diets andweight gains were determined at 21 days. Triangles: flour from corn seedcontaining vector pNOV4057. Diamonds: flour from corn seed containingvector pNOV4061.

FIG. 8B: Phytase formulated as milled corn was added to low phosphatechicken feed before steam conditioning and pelleting. Squares: maizeflour from corn seed containing pNOV4061. Diamonds: maize flour fromcorn seed containing pNOV4057.

FIG. 8C: Phytase formulated as milled corn was added to low phosphatechicken feed before steam conditioning and pelleting. Corn seedcontaining phytase (encoded by vector pNOV4057) was milled to differentaverage particle sizes ranging from a fine flour to a coarse grist.Diamonds, fine grind (flour); squares, medium grind, triangles, coarsegrind. Coarse grind material consisted predominantly of particles >2000microns. Medium grind material was predominantly in the size range of500-2000 microns. Fine grind was <500 microns.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

“Altered levels” refers to the level of expression in transformed ortransgenic cells or organisms that differs from that of normal oruntransformed cells or organisms.

The term “altered plant trait” means any phenotypic or genotypic changein a transgenic plant relative to the wild-type, non-transformed ornon-transgenic plant host.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Chimeric” is used to indicate that a DNA sequence, such as a vector ora gene, is comprised of more than one DNA sequences of distinct originwhich are fused together by recombinant DNA techniques resulting in aDNA sequence, which does not occur naturally. The term “chimeric gene”refers to any gene that contains 1) DNA sequences, including regulatoryand coding sequences, that are not found together in nature, or 2)sequences encoding parts of proteins not naturally adjoined, or 3) partsof promoters that are not naturally adjoined. Accordingly, a chimericgene may comprise regulatory sequences and coding sequences that arederived from different sources, or comprise regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent from that found in nature.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that provideresistance to antibiotics such as tetracycline, hygromycin orampicillin, or other means for selection of transformed cells.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Constitutive promoter” refers to a promoter that is able to express thegene that it controls in all or nearly all of the plant tissues duringall or nearly all developmental stages of the plant. Each of thetranscription-activating elements do not exhibit an absolutetissue-specificity, but mediate transcriptional activation in most plantparts at a level of ≧1% of the level reached in the part of the plant inwhich transcription is most active.

The term “contacting” may include any method known or described forintroducing a nucleic acid segment into a cell.

“Expression” refers to the transcription and/or translation of anendogenous gene or a transgene in plants. For example, in the case ofantisense constructs, expression may refer to the transcription of theantisense DNA only. In addition, expression refers to the transcriptionand stable accumulation of sense (mRNA) or functional RNA. Expressionmay also refer to the production of protein.

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassettecomprising the nucleotide sequence of interest may be chimeric, meaningthat at least one of its components is heterologous with respect to atleast one of its other components. The expression cassette may also beone which is naturally occurring but has been obtained in a recombinantform useful for heterologous expression. The expression of thenucleotide sequence in the expression cassette may be under the controlof a constitutive promoter or of an inducible promoter which initiatestranscription only when the host cell is exposed to some particularexternal stimulus. In the case of a multicellular organism, the promotercan also be specific to a particular tissue or organ or stage ofdevelopment.

The “expression pattern” of a promoter (with or without enhancer) is thepattern of expression levels which shows where in the plant and in whatdevelopmental stage transcription is initiated by the promoter.Expression patterns of a set of promoters are said to be complementarywhen the expression pattern of one promoter shows little overlap withthe expression pattern of the other promoter. The level of expression ofa promoter can be determined by measuring the ‘steady state’concentration of a standard transcribed reporter mRNA. This measurementis indirect since the concentration of the reporter mRNA is dependentnot only on its synthesis rate, but also on the rate with which the mRNAis degraded. Therefore the steady state level is the product ofsynthesis rates and degradation rates.

The rate of degradation can however be considered to proceed at a fixedrate when the transcribed sequences are identical, and thus this valuecan serve as a measure of synthesis rates. When promoters are comparedin this way techniques available to those skilled in the art arehybridization S1-RNAse analysis. Northern blots and competitive RT-PCR.This list of techniques in no way represents all available techniques,but rather describes commonly used procedures used to analyzetranscription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promotershas revealed that there is usually no single base at which transcriptionstarts, but rather a more or less clustered set of initiation sites,each of which accounts for some start points of the mRNA. Since thisdistribution varies from promoter to promoter the sequences of thereporter mRNA in each of the populations would differ from each other.Since each mRNA species is more or less prone to degradation, no singledegradation rate can be expected for different reporter mRNAs. It hasbeen shown for various eukaryotic promoter sequences that the sequencesurrounding the initiation site (‘initiator’) plays an important role indetermining the level of RNA expression directed by that specificpromoter. This includes also part of the transcribed sequences. Thedirect fusion of promoter to reporter sequences would therefore lead tosuboptimal levels of transcription.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al., 1995).

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNA,or specific protein, including regulatory sequences. Genes also includenonexpressed DNA segments that, for example, form recognition sequencesfor other proteins. Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information, and may include sequences designed tohave desired parameters.

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the plant and stablyinherited by progeny through successive generations.

“Genome” refers to the complete genetic material of an organism.

“Germline cells” refer to cells that are destined to be gametes andwhose genetic material is heritable.

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous polynucleic acid,” as used herein, each refer to asequence that originates from a source foreign to the particular hostcell or, if from the same source, is modified from its original form.Thus, a heterologous gene in a host cell includes a gene that isendogenous to the particular host cell but has been modified through,for example, the use of DNA shuffling. The terms also includenon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

“Inducible promoter” refers to those regulated promoters that can beturned on in one or more cell types by an external stimulus, such as achemical, light, hormone, stress, or a pathogen.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

The term “intracellular localization sequence” refers to a nucleotidesequence that encodes an intracellular targeting signal. An“intracellular targeting signal” is an amino acid sequence that istranslated in conjunction with a protein and directs it to a particularsub-cellular compartment. “Endoplasmic reticulum (ER) stop transitsignal” refers to a carboxy-terminal extension of a polypeptide, whichis translated in conjunction with the polypeptide and causes a proteinthat enters the secretory pathway to be retained in the ER. “ER stoptransit sequence” refers to a nucleotide sequence that encodes the ERtargeting signal. Other intracellular targeting sequences encodetargeting signals active in seeds and/or leaves and vacuolar targetingsignals.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” polynucleic acid (polynucleotide) segment oran “isolated” or “purified” polypeptide is a polynucleic acid segment orpolypeptide that, by the hand of man, exists apart from its nativeenvironment and is therefore not a product of nature. An isolatedpolynucleic acid segment or polypeptide may exist in a purified form ormay exist in a non-native environment such as, for example, a transgenichost cell. For example, an “isolated” or “purified” polynucleic acidsegment or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” polynucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived. Aprotein that is substantially free of cellular material includespreparations of protein or polypeptide having less than about 30%, 20%,10%, 5%, (by dry weight) of contaminating protein. When the protein ofthe invention, or biologically active fragment (e.g., catalytically)thereof, is recombinantly produced, preferably culture medium representsless than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals. Fragments and variantsof the disclosed nucleotide sequences and proteins or partial-lengthproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence, and hence a portion of thepolypeptide or protein, encoded thereby.

A “marker gene” encodes a selectable or screenable trait.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

The term “native gene” refers to gene that is present in the genome ofan untransformed cell.

“Naturally occurring” is used to describe an object that can be found innature as distinct from being artificially produced by man. For example,a protein or nucleotide sequence present in an organism (including avirus), which can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory, is naturallyoccurring.

Nov9X and Nov9x, are used interchangeably.

The term “polynucleotide”, “nucleic acid”, “polynucleic acid” or“polynucleic acid segment” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985;Rossolini et al., 1994).

A “nucleic acid fragment” is a fraction of a given nucleic acidmolecule. In higher plants, deoxyribonucleic acid (DNA) is the geneticmaterial while ribonucleic acid (RNA) is involved in the transfer ofinformation contained within DNA into proteins. A “genome” is the entirebody of genetic material contained in each cell of an organism. The term“nucleotide sequence” refers to a polymer of DNA or RNA which can besingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases capable of incorporation into DNA or RNApolymers. The terms “nucleic acid” or “nucleic acid sequence” may alsobe used interchangeably with gene, cDNA, DNA and RNA encoded by a gene(Batzer et al., 1991; Ohtsuka et al., 1985; Rossolini et al., 1999).

Expression cassettes employed to introduce a phytase encoding openreading frame of the invention to a host cell preferably comprise atranscriptional initiation region linked to the open reading frame. Suchan expression cassette may be provided with a plurality of restrictionsites for insertion of the open reading frame and/or other DNAs, e.g., atranscriptional regulatory regions and/or selectable marker gene(s).

The transcriptional cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region,the DNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions for plant cells are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also, Guerineau et al., 1991;Proudfoot, 1991; Sanfacon et al., 1991; Mogen et al., 1990; Munroe etal., 1990; Ballas et al., 1989; Joshi et al., 1987.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

“Operably linked” when used with respect to nucleic acid, means joinedas part of the same nucleic acid molecule, suitably positioned andoriented for transcription to be initiated from the promoter. DNAoperably linked to a promoter is “under transcriptional initiationregulation” of the promoter. Coding sequences can be operably-linked toregulatory sequences in sense or antisense orientation. When used withrespect to polypeptides, “operably linked” means joined as part of thesame polypeptide, i.e., via peptidyl bonds.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed cells or organisms.

Known methods of polymerase chain reaction “PCR” include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like. Seealso Innis et al., 1995; and Gelfand, 1995; and Innis and Gelfand, 1999.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

“Primary transformant” and “T0 generation” refer to transgenic plantsthat are of the same genetic generation as the tissue which wasinitially transformed (i.e., not having gone through meiosis andfertilization since transformation).

“Production tissue” refers to mature, harvestable tissue consisting ofnon-dividing, terminally-differentiated cells. It excludes young,growing tissue consisting of germline, meristematic, andnot-fully-differentiated cells.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. It is capable ofoperating in both orientations (normal or flipped), and is capable offunctioning even when moved either upstream or downstream from thepromoter. Both enhancers and other upstream promoter elements bindsequence-specific DNA-binding proteins that mediate their effects.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even be comprised of synthetic DNA segments. A promoter mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor or factors, the minimalpromoter functions to permit transcription. A “minimal or core promoter”thus consists only of all basal elements needed for transcriptioninitiation, e.g., a TATA box and/or an initiator.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and include both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. New promoters of various types useful in plantcells are constantly being discovered, numerous examples may be found inthe compilation by Okamuro et al. (1989). Typical regulated promotersuseful in plants include but are not limited to safener-induciblepromoters, promoters derived from the tetracycline-inducible system,promoters derived from salicylate-inducible systems, promoters derivedfrom alcohol-inducible systems, promoters derived fromglucocorticoid-inducible system, promoters derived frompathogen-inducible systems, and promoters derived fromecdysome-inducible systems.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences which may be a combination of syntheticand natural sequences. As is noted above, the term “suitable regulatorysequences” is not limited to promoters. Some suitable regulatorysequences useful with plants in the present invention will include, butare not limited to constitutive plant promoters, plant tissue-specificpromoters, plant development-specific promoters, inducible plantpromoters and viral promoters.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Secondary transformants” and the “T1, T2, T3, etc. generations” referto transgenic plants derived from primary transformants through one ormore meiotic and fertilization cycles. They may be derived byself-fertilization of primary or secondary transformants or crosses ofprimary or secondary transformants with other transformed oruntransformed plants.

“Specific expression” is the expression of gene products which islimited to one or a few plant tissues (spatial limitation) and/or to oneor a few plant developmental stages (temporal limitation). It isacknowledged that hardly a true specificity exists: promoters seem to bepreferably switch on in some tissues, while in other tissues there canbe no or only little activity. This phenomenon is known as leakyexpression. However, with specific expression in this invention is meantpreferable expression in one or a few plant tissues.

“Stably transformed” refers to cells that have been selected andregenerated on a selection media following transformation.

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989).

“Tissue-specific promoter” refers to regulated promoters that are notexpressed in all cells of an organism, e.g., not in all plant cells, butonly in one or more cell types in specific organs (such as leaves orseeds), specific tissues (such as embryo or cotyledon), or specific celltypes (such as leaf parenchyma or seed storage cells). These alsoinclude promoters that are temporally regulated, such as in early orlate embryogenesis, during fruit ripening in developing seeds or fruit,in fully differentiated leaf, or at the onset of senescence.

“Transcription Stop Fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples include the 3′non-regulatory regions of genes encoding nopaline synthase and the smallsubunit of ribulose bisphosphate carboxylase.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.Examples of methods of transformation of plants and plant cells includeAgrobacterium-mediated transformation (De Blaere et al., 1987) andparticle bombardment technology (Klein et al., 1987; U.S. Pat. No.4,945,050), however, many other methods of transformation of cells areknown to the art. Whole plants may be regenerated from transgenic cellsby methods well known to the skilled artisan (see, for example, Fromm etal., 1990).

“Transformed,” “transgenic,” and “recombinant” refer to a host organismsuch as a plant into which a heterologous nucleic acid molecule has beenintroduced. The nucleic acid molecule can be stably integrated into thegenome by methods generally known in the art which are disclosed inSambrook et al., 1989). For example, “transformed,” “transformant,” and“transgenic” plants or calli have been through the transformationprocess and contain a foreign gene integrated into their chromosome. Theterm “untransformed” refers to normal plants that have not been throughthe transformation process.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, genes that are either heterologous or homologous to the genesof a particular plant to be transformed. Additionally, transgenes maycomprise native genes inserted into a non-native organism, or chimericgenes. The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism but that is introduced by genetransfer.

A “transgenic plant” is a plant having one or more plant cells thatcontain a heterologous DNA sequence.

“Transient expression” refers to expression in cells in which atransgene is introduced, e.g., by viral infection or by such methods asAgrobacterium-mediated transformation, electroporation, or biolisticbombardment, but is not selected for its stable maintenance.

“Transiently transformed” refers to cells in which an expressioncassette, polynucleotide or transgene has been introduced (for example,by such methods as Agrobacterium-mediated transformation or biolisticbombardment), but not selected for stable maintenance.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

“Translation Stop Fragment” refers to nucleotide sequences that containone or more regulatory signals, such as one or more termination codonsin all three frames, capable of terminating translation. Insertion of atranslation stop fragment adjacent to or near the initiation codon atthe 5′ end of the coding sequence will result in no translation orimproper translation. Excision of the translation stop fragment bysite-specific recombination will leave a site-specific sequence in thecoding sequence that does not interfere with proper translation usingthe initiation codon.

A polypeptide or enzyme exhibiting “phytase” activity or a “phytase” isintended to cover any enzyme capable of effecting the liberation ofinorganic phosphate or phosphorous from various myo-inositol phosphates.Examples of such myo-inositol phosphates (phytase substrates) are phyticacid and any salt thereof, e.g., sodium phytate or potassium phytate ormixed salts. Also any stereoisomer of the mono-, di-, tri-, tetra- orpenta-phosphates of myo-inositol may serve as a phytase substrate. Inaccordance with the above definition, the phytase activity can bedetermined using any assay in which one of these substrates is used.

A thermotolerant phytase of the invention includes variant polypeptidesderived from a particular thermotolerant phytase by deletion (so-calledtruncation) or addition of one or more amino acids to the N-terminaland/or C-terminal end of the native protein; deletion or addition of oneor more amino acids at one or more sites in the native protein; orsubstitution of one or more amino acids at one or more sites in thethermotolerant phytase. Such variants may result from, for example, fromhuman manipulation. Methods for such manipulations are generally knownin the art. For example, amino acid sequence variants of thepolypeptides can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel, 1985; Kunkel et al., 1987; U.S. Pat. No.4,873,192; Walker and Gaastra, 1983, and the references cited therein.Guidance as to appropriate amino acid substitutions that do not affectbiological activity of the protein of interest may be found in the modelof Dayhoff et al., 1978, herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, are preferred.

Thus, the thermotolerant phytase genes and nucleotide sequences of theinvention include both the naturally occurring sequences as well asmutant forms. Likewise, the thermotolerant phytase polypeptides of theinvention encompass both naturally occurring proteins as well asvariations and modified forms thereof. Such variants will continue topossess the desired activity. The deletions, insertions, andsubstitutions of the polypeptide sequence encompassed herein are notexpected to produce radical changes in the characteristics of thepolypeptide. However, when it is difficult to predict the exact effectof the substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays.

The nucleic acid molecules of the invention are optimized for enhancedexpression in an organism of interest. For plants, see, for example,EPA035472; WO 91/16432; Perlak et al., 1991; and Murray et al., 1989. Inthis manner, the genes or gene fragments can be synthesized utilizingplant-preferred codons. See, for example, Campbell and Gowri, 1990 for adiscussion of host-preferred codon usage. It is recognized that all orany part of the gene sequence may be optimized or synthetic. That is,synthetic or partially optimized sequences may also be used. Variantnucleotide sequences and proteins also encompass sequences and proteinderived from a mutagenic and recombinogenic procedure such as DNAshuffling. With such a procedure, one or more different coding sequencescan be manipulated to create a new polypeptide possessing the desiredproperties. In this manner, libraries of recombinant polynucleotides aregenerated from a population of related sequence polynucleotidescomprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. Strategies for suchDNA shuffling are known in the art. See, for example, Stemmer, 1994;Stemmer, 1994; Crameri et al., 1997; Moore et al., 1997; Zhang et al.,1997; Crameri et al., 1998; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

By “variants” is intended substantially similar sequences. Fornucleotide sequences, variants include those sequences that, because ofthe degeneracy of the genetic code, encode the identical amino acidsequence of the reference protein. Naturally occurring allelic variantssuch as these can be identified with the use of well-known molecularbiology techniques, as, for example, with polymerase chain reaction(PCR) and hybridization techniques. Variant nucleotide sequences alsoinclude synthetically derived nucleotide sequences, such as thosegenerated, for example, by using site-directed mutagenesis which encodethe reference protein, as well as those that encode a polypeptide havingamino acid substitutions. Generally, nucleotide sequence variants of theinvention will have at least 40%, 50%, 60%, preferably 70%, morepreferably 80%, even more preferably 90%, most preferably 99%, andsingle unit percentage identity to the native nucleotide sequence basedon these classes. For example, 71%, 72%, 73% and the like, up to atleast the 90% class. Variants may also include a full length genecorresponding to an identified gene fragment.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Preferred Constructs of the Invention and Host Cells

The invention preferably provides an expression cassette which comprisesa nucleic acid sequence (promoter) capable of directing expression of apolynucleotide encoding a thermotolerant phytase either in vitro or invivo. As described hereinbelow, preferred polynucleotides of theinvention are optimized for expression in a particular organism, e.g., aplant. Methods to prepare and/or identify a thermotolerant phytaseinclude mutagenesis, e.g., recursive mutagenesis, and/or selection orscreening, e.g., for phytases having activity at temperatures greaterthan 60° C. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art. See, for example, Kunkel, 1985; Kunkel etal., 1987; U.S. Pat. No. 4,873,192; Walker and Gaastra, 1983 and thereferences cited therein and Arnold et al., 1996). Once a polynucleotideencoding a thermotolerant phytase is identified, the sequence of thepolynucleotide may be optimized. Methods to optimize the expression of anucleic acid segment in a particular organism are well known in the art.Briefly, a codon usage table indicating the optimal codons used by thetarget organism is obtained and optimal codons are selected to replacethose in the target polynucleotide and the optimized sequence is thenchemically synthesized. Preferred codons for maize are described in U.S.Pat. No. 5,625,136.

DNA and Host Cells for Transformation

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) BACs(bacterial artificial chromosomes) and DNA segments for use intransforming cells will generally comprise the phytase encoding DNA, aswell as other DNA such as cDNA, gene or genes which one desires tointroduce into the cells. These DNA constructs can further includestructures such as promoters, enhancers, polylinkers, or even regulatorygenes as desired. One of the DNA segments or genes chosen for cellularintroduction will often encode a protein which will be expressed in theresultant transformed (recombinant) cells, such as will result in ascreenable or selectable trait and/or which will impart an improvedphenotype to the transformed cell or plant regenerated from atransformed plant cell. However, this may not always be the case, andthe present invention also encompasses transformed cells and plantsincorporating non-expressed transgenes.

DNA useful for introduction into cells includes that which has beenderived or isolated from any source, that may be subsequentlycharacterized as to structure, size and/or function, chemically altered,and later introduced into plants. An example of DNA “derived” from asource, would be a DNA sequence that is identified as a useful fragmentwithin a given organism, and which is then chemically synthesized inessentially pure form. An example of such DNA “isolated” from a sourcewould be a useful DNA sequence that is excised or removed from saidsource by chemical means, e.g., by the use of restriction endonucleases,so that it can be further manipulated, e.g., amplified, for use in theinvention, by the methodology of genetic engineering. Such DNA iscommonly referred to as “recombinant DNA.”

Therefore useful DNA includes completely synthetic DNA, semi-syntheticDNA, DNA isolated from biological sources, and DNA derived fromintroduced RNA. Generally, the introduced DNA is not originally residentin the genotype which is the recipient of the DNA, but it is within thescope of the invention to isolate a gene from a given genotype, and tosubsequently introduce multiple copies of the gene into the samegenotype, e.g., to enhance production of a given gene product.

The introduced DNA includes, but is not limited to, DNA from plantgenes, and non-plant genes such as those from bacteria, yeasts, fungi,animals or viruses. The introduced DNA can include modified or syntheticgenes, portions of genes, or chimeric genes, including genes from thesame or different genotype. The term “chimeric gene” or “chimeric DNA”is defined as a gene or DNA sequence or segment comprising at least twoDNA sequences or segments from species which do not combine DNA undernatural conditions, or which DNA sequences or segments are positioned orlinked in a manner which does not normally occur in the native genome ofthe untransformed cell.

The introduced DNA used for transformation herein may be circular orlinear, double-stranded or single-stranded. Generally, the DNA is in theform of chimeric DNA, such as plasmid DNA, that can also contain codingregions flanked by regulatory sequences which promote the expression ofthe recombinant DNA present in the transformed cell. For example, theDNA may itself comprise or consist of a promoter that is active in acell which is derived from a source other than that cell, or may utilizea promoter already present in the cell that is the transformationtarget.

Generally, the introduced DNA will be relatively small, i.e., less thanabout 30 kb to minimize any susceptibility to physical, chemical, orenzymatic degradation which is known to increase as the size of the DNAincreases. The number of proteins, RNA transcripts or mixtures thereofwhich is introduced into the cell is preferably preselected and defined,e.g., from one to about 5-10 such products of the introduced DNA may beformed.

The selection of an appropriate expression vector will depend upon thehost cells. Typically an expression vector contains (1) prokaryotic DNAelements coding for a bacterial origin of replication and an antibioticresistance gene to provide for the amplification and selection of theexpression vector in a bacterial host; (2) DNA elements that controlinitiation of transcription such as a promoter; (3) DNA elements thatcontrol the processing of transcripts such as introns, transcriptiontermination/polyadenylation sequence; and (4) a gene of interest that isoperatively linked to the DNA elements to control transcriptioninitiation. The expression vector used may be one capable ofautonomously replicating in the above host or capable of integratinginto the chromosome, originally containing a promoter at a site enablingtranscription of the linked phytase gene.

If prokaryotes such as bacteria are used as the host, the expressionvector for the phytase is preferably one capable of autonomouslyreplicating in the micro-organism and comprising a promoter, aribosome-binding sequence, the novel phytase gene, and a transcriptiontermination sequence. The vector may also contain a gene for regulatingthe promoter.

A general descriptions of plant expression vectors and reporter genescan be found in Gruber et al. (1993).

Mammalian expression vectors may comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences.

Suitable vectors include by way of example: for bacteria, pQE70, pQE60,pQE-9 (Qiagen), pBluescript II (Stratagene), pTRC99a, pKK223-3, pDR540,pRIT2T (Pharmacia); for eukaryotic cells: pXT1, pSG5 (Stratagene) pSVK3,pBPV, pMSG, pSVLSV40 (Pharmacia). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1(Promega Biotec, Madison, Wis., USA). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

In certain embodiments, it is contemplated that one may wish to employreplication-competent viral vectors in monocot transformation. Suchvectors include, for example, wheat dwarf virus (WDV) “shuttle” vectors,such as pW1-11 and PW1-GUS (Ugaki et al., 1991). These vectors arecapable of autonomous replication in maize cells as well as E. coli, andas such may provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It is also contemplated that transposable elements would beuseful for introducing DNA fragments lacking elements necessary forselection and maintenance of the plasmid vector in bacteria, e.g.,antibiotic resistance genes and origins of DNA replication. It is alsoproposed that use of a transposable element such as Ac, Ds, or Mu wouldactively promote integration of the desired DNA and hence increase thefrequency of stably transformed cells.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis; andvarious species within the genera Escherichia, Pseudomonas, Serratia,Streptomyces, Corynebacterium, Brevibacterium, Bacillus, Microbacterium,and Staphylococcus, although others may also be employed as a matter ofchoice; fungal cells belonging to the genera Aspergillus, Rhizopus,Trichoderma, Neurospora, Mucor, Penicillium, etc., such as yeastbelonging to the genera Kluyveromyces, Saccharomyces,Schizosaccharomyces, Trichosporon, Schwanniomyces, etc.; insect cellssuch as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COSor Bowes melanoma, C127, 3T3, CHO, HeLa and BHK cell lines; plant cells,and the like. Any host can be used insofar as it can express the gene ofinterest.

The construction of vectors which may be employed in conjunction withthe present invention will be known to those of skill of the art inlight of the present disclosure (see, e.g., Sambrook et al., 1989;Gelvin et al., 1990).

The expression cassette of the invention may contain one or a pluralityof restriction sites allowing for placement of the polynucleotideencoding a thermotolerant phytase under the regulation of a regulatorysequence. The expression cassette may also contain a termination signaloperably linked to the polynucleotide as well as regulatory sequencesrequired for proper translation of the polynucleotide. The expressioncassette containing the polynucleotide of the invention may be chimeric,meaning that at least one of its components is heterologous with respectto at least one of the other components. Expression of thepolynucleotide in the expression cassette may be under the control of aconstitutive promoter, inducible promoter, regulated promoter,tissue-specific promoter, viral promoter or synthetic promoter.

The expression cassette may include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region,the polynucleotide of the invention and a transcriptional andtranslational termination region functional in vivo and/or in vitro. Thetermination region may be native with the transcriptional initiationregion, may be native with the polynucleotide, or may be derived fromanother source.

The regulatory sequences may be located upstream (5′ non-codingsequences), within (intron), or downstream (3′ non-coding sequences) ofa coding sequence, and influence the transcription, RNA processing orstability, and/or translation of the associated coding sequence.Regulatory sequences may include, but are not limited to, enhancers,promoters, repressor binding sites, translation leader sequences,introns, and polyadenylation signal sequences. They may include naturaland synthetic sequences as well as sequences which may be a combinationof synthetic and natural sequences.

The vector, used in the present invention may also include appropriatesequences for amplifying expression.

Regulatory Sequences

A promoter is a nucleotide sequence which controls the expression of acoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. A promoter includes aminimal promoter, consisting only of all basal elements needed fortranscription initiation, such as a TATA-box and/or initiator that is ashort DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. A promoter maybe derived entirely from a native gene, or be composed of differentelements derived from different promoters found in nature, or even becomprised of synthetic DNA segments. A promoter may contain DNAsequences that are involved in the binding of protein factors whichcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions. A promoter may also include aminimal promoter plus a regulatory element or elements capable ofcontrolling the expression of a coding sequence or functional RNA. Thistype of promoter sequence contains of proximal and more distal elements,the latter elements are often referred to as enhancers.

Representative examples of promoters include, but are not limited to,promoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. Particular bacterial promotersinclude E. coli lac or trp, the phage lambda P_(L) promoter, lacI, lacZ,T3, T7; gpt, and lambda P_(R). Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-I. Also, an enhancer for the IEgene from human CMV may be used together with the promoter.

Any promoter capable of expressing in yeast hosts can be used as thepromoter. Examples thereof include promoters for genes of hexokinase andthe like in the glycolytic pathway, and promoters such as gal 1promoter, gal 10 promoter, heat shock protein promoter, MFα-1 promoterand CUP 1 promoter.

Any promoter capable of expressing in filamentous fungi may be used.Examples are a promoter induced strongly by starch or cellulose, e.g., apromoter for glucoamylase or α-amylase from the genus Aspergillus orcellulase (cellobiohydrase) from the genus Trichoderma, a promoter forenzymes in the glycolytic pathway, such as phosphoglycerate kinase (pgk)and glycerylaldehyde 3-phosphate dehydrogenase (gpd), etc.

Within a plant promoter region there are several domains that arenecessary for full function of the promoter. The first of these domainslies immediately upstream of the structural gene and forms the “corepromoter region” containing consensus sequences, normally 70 base pairsimmediately upstream of the gene. The core promoter region contains thecharacteristic CAAT and TATA boxes plus surrounding sequences, andrepresents a transcription initiation sequence that defines thetranscription start point for the structural gene.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional.Furthermore, the core promoter region is insufficient to provide fullpromoter activity. A series of regulatory sequences upstream of the coreconstitute the remainder of the promoter. The regulatory sequencesdetermine expression level, the spatial and temporal pattern ofexpression and, for an important subset of promoters, expression underinductive conditions (regulation by external factors such as light,temperature, chemicals, hormones).

A range of naturally-occurring promoters are known to be operative inplants and have been used to drive the expression of heterologous (bothforeign and endogenous) genes in plants: for example, the constitutive35S cauliflower mosaic virus (CaMV) promoter, the ripening-enhancedtomato polygalacturonase promoter (Bird et al., 1988), the E8 promoter(Diekman & Fischer, 1988) and the fruit specific 2A1 promoter (Pear etal., 1989) and many others.

Two principal methods for the control of expression are known, viz.:overexpression and underexpression. Overexpression can be achieved byinsertion of one or more than one extra copy of the selected gene. Itis, however, not unknown for plants or their progeny, originallytransformed with one or more than one extra copy of a nucleotidesequence, to exhibit the effects of underexpression as well asoverexpression. For underexpression there are two principle methodswhich are commonly referred to in the art as “antisense downregulation”and “sense downregulation” (sense downregulation is also referred to as“cosuppression”). Generically these processes are referred to as “genesilencing”. Both of these methods lead to an inhibition of expression ofthe target gene.

Obtaining sufficient levels of transgene expression in the appropriateplant tissues is an important aspect in the production of geneticallyengineered crops. Expression of heterologous DNA sequences in a planthost is dependent upon the presence of an operably linked promoter thatis functional within the plant host. Choice of the promoter sequencewill determine when and where within the organism the heterologous DNAsequence is expressed.

Therefore, the selection of promoters for directing expression of agiven transgene is critical. Promoters which are useful for planttransgene expression include those that are inducible, viral, synthetic,constitutive (Odell et al., 1985), temporally regulated, spatiallyregulated, tissue-specific, and spatio-temporally regulated.

Where expression in specific tissues or organs is desired,tissue-specific promoters may be used. In contrast, where geneexpression in response to a stimulus is desired, inducible promoters arethe regulatory elements of choice. Where continuous expression isdesired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in expression constructsof transformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

A number of plant promoters have been described with various expressioncharacteristics. Examples of some constitutive promoters which have beendescribed include the rice actin 1 (Wang et al., 1992; U.S. Pat. No.5,641,876), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al.,1987), sucrose synthase, and the ubiquitin promoters.

Examples of tissue specific promoters which have been described includethe lectin (Vodkin, 1983; Lindstrom et al., 1990), corn alcoholdehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn lightharvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shockprotein (Odell et al., 1985), pea small subunit RuBP carboxylase(Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopinesynthase (Langridge et al., 1989), Ti plasmid nopaline synthase(Langridge et al., 1989), petunia chalcone isomerase (vanTunen et al.,1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV35S (Odell et al., 1985), potato patatin (Wenzler et al., 1989), rootcell (Yamamoto et al., 1990), maize zein (Reina et al., 1990; Kriz etal., 1987; Wandelt et al., 1989; Langridge et al., 1983; Reina et al.,1990), globulin-1 (Belanger et al., 1991), α-tubulin, cab (Sullivan etal., 1989), PEPCase (Hudspeth & Grula, 1989), R gene complex-associatedpromoters (Chandler et al., 1989), and chalcone synthase promoters(Franken et al., 1991).

Inducible promoters that have been described include the ABA- andturgor-inducible promoters, the promoter of the auxin-binding proteingene (Schwob et al., 1993), the UDP glucose flavonoidglycosyl-transferase gene promoter (Ralston et al., 1988), the MPIproteinase inhibitor promoter (Cordero et al., 1994), and theglyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al.,1995; Quigley et al., 1989; Martinez et al., 1989).

Several tissue-specific regulated genes and/or promoters have beenreported in plants. These include genes encoding the seed storageproteins (such as napin, cruciferin, beta-conglycinin, and phaseolin)zein or oil body proteins (such as oleosin), or genes involved in fattyacid biosynthesis (including acyl carrier protein, stearoyl-ACPdesaturase. And fatty acid desaturases (fad 2-1)), and other genesexpressed during embryo development (such as Bce4, see, for example. EP255378 and Kridl et al., 1991). Particularly useful for seed-specificexpression is the pea vicilin promoter (Czako et al., 1992). (See alsoU.S. Pat. No. 5,625,136, herein incorporated by reference.) Other usefulpromoters for expression in mature leaves are those that are switched onat the onset of senescence, such as the SAG promoter from Arabidopsis(Gan et al., 1995).

A class of fruit-specific promoters expressed at or during antithesisthrough fruit development, at least until the beginning of ripening, isdiscussed in U.S. Pat. No. 4,943,674, the disclosure of which is herebyincorporated by reference. CDNA clones that are preferentially expressedin cotton fiber have been isolated (John et al., 1992). CDNA clones fromtomato displaying differential expression during fruit development havebeen isolated and characterized (Mansson et al., 1985, Slater et al.,1985). The promoter for polygalacturonase gene is active in fruitripening. The polygalacturonase gene is described in U.S. Pat. No.4,535,060, U.S. Pat. No. 4,769,061, U.S. Pat. No. 4,801,590, and U.S.Pat. No. 5,107,065, which disclosures are incorporated herein byreference.

Other examples of tissue-specific promoters include those that directexpression in leaf cells following damage to the leaf (for example, fromchewing insects), in tubers (for example, patatin gene promoter), and infiber cells (an example of a developmentally-regulated fiber cellprotein is E6 (John et al., 1992). The E6 gene is most active in fiber,although low levels of transcripts are found in leaf, ovule and flower.

The tissue-specificity of some “tissue-specific” promoters may not beabsolute and may be tested by one skilled in the art using thediphtheria toxin sequence. One can also achieve tissue-specificexpression with “leaky” expression by a combination of differenttissue-specific promoters (Beals et al., 1997). Other tissue-specificpromoters can be isolated by one skilled in the art (see U.S. Pat. No.5,589,379).

Ultimately, the most desirable DNA segments for introduction into amonocot genome may be homologous genes or gene families which encode adesired trait (e.g., hydrolysis of proteins, lipids or polysaccharides)and which are introduced under the control of novel promoters orenhancers, etc., or perhaps even homologous or tissue specific (e.g.,root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- orleaf-specific) promoters or control elements. Indeed, it is envisionedthat a particular use of the present invention will be the targeting ofa gene in a constitutive manner or in an inducible manner.

Vectors useful for use in tissue-specific targeting of genes intransgenic plants typically include tissue-specific promoters and mayalso include other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure. These include, for example, the rbcSpromoter, specific for green tissue; the ocs, nos and mas promoterswhich have higher activity in roots or wounded leaf tissue; a truncated(−90 to +8) 35S promoter which directs enhanced expression in roots, anα-tubulin gene that directs expression in roots and promoters derivedfrom zein storage protein genes which direct expression in endosperm.

Tissue specific expression may be functionally accomplished byintroducing a constitutively expressed gene (all tissues) in combinationwith an antisense gene that is expressed only in those tissues where thegene product is not desired. For example, a gene coding for a lipase maybe introduced such that it is expressed in all tissues using the 35Spromoter from Cauliflower Mosaic Virus. Expression of an antisensetranscript of the lipase gene in a maize kernel, using for example azein promoter, would prevent accumulation of the lipase protein in seed.Hence the protein encoded by the introduced gene would be present in alltissues except the kernel.

Expression of a gene in a transgenic plant may be desired only in acertain time period during the development of the plant. Developmentaltiming is frequently correlated with tissue specific gene expression.For example, expression of zein storage proteins is initiated in theendosperm about 15 days after pollination.

Several inducible promoters are known in the art. Many are described ina review by Gatz (1996) (see also Gatz, 1997). Examples includetetracycline repressor system, Lac repressor system, copper-induciblesystems, salicylate-inducible systems (such as the PR1a system),glucocorticoid-inducible (Aoyama T. et al., 1997) and ecdysome-induciblesystems. Also included are the benzene sulphonamide-inducible (U.S. Pat.No. 5,364,780) and alcohol-inducible (WO 97/06269 and WO 97/06268)inducible systems and glutathione S-transferase promoters. In the caseof a multicellular organism, the promoter can also be specific to aparticular tissue, organ or stage of development. Examples of suchpromoters include, but are not limited to, the Zea mays ADP-gpp and theZea mays γ-zein promoter.

Other studies have focused on genes inducibly regulated in response toenvironmental stress or stimuli such as increased salinity. Drought,pathogen and wounding (Graham et al., 1985; Graham et al., 1985, Smithet al., 1986). Accumulation of metallocarboxypeptidase-inhibitor proteinhas been reported in leaves of wounded potato plants (Graham et al.,1981). Other plant genes have been reported to be induced methyljasmonate, elicitors, heat-shock, anaerobic stress, or herbicidesafeners.

Regulated expression of a chimeric transacting viral replication proteincan be further regulated by other genetic strategies. For example,Cre-mediated gene activation as described by Odell et al. 1990. Thus, aDNA fragment containing 3′ regulatory sequence bound by lox sitesbetween the promoter and the replication protein coding sequence thatblocks the expression of a chimeric replication gene from the promotercan be removed by Cre-mediated excision and result in the expression ofthe trans-acting replication gene. In this case, the chimeric Cre gene,the chimeric trans-acting replication gene, or both can be under thecontrol of tissue- and developmental-specific or inducible promoters. Analternate genetic strategy is the use of tRNA suppressor gene. Forexample, the regulated expression of a tRNA suppressor gene canconditionally control expression of a trans-acting replication proteincoding sequence containing an appropriate termination codon as describedby Ulmasov et al., 1997. Again, either the chimeric tRNA suppressorgene, the chimeric transacting replication gene, or both can be underthe control of tissue- and developmental-specific or induciblepromoters.

In addition to the use of a particular promoter, other types of elementscan influence expression of transgenes. In particular, introns havedemonstrated the potential for enhancing transgene expression. Forexample, Callis et al. (1987) described an intron from the corn alcoholdehydrogenase gene, which is capable of enhancing the expression oftransgenes in transgenic plant cells. Similarly, Vasil et al. (1989)described an intron from the corn sucrose synthase gene having similarenhancing activity. The rice actin 1 intron, has been widely used in theenhancement of transgene expression in a number of different transgeniccrops. (McElroy et al., 1991).

Other elements include those that can be regulated by endogenous orexogenous agents, e.g., by zinc finger proteins, including naturallyoccurring zinc finger proteins or chimeric zinc finger proteins. See,e.g., U.S. Pat. No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311.

An enhancer is a DNA sequence which can stimulate promoter activity andmay be an innate element of the promoter or a heterologous elementinserted to enhance the level or tissue specificity of a particularpromoter. An enhancer is capable of operating in both orientations (5′to 3′ and 3′-5′ relative to the gene of interest coding sequences), andis capable of functioning even when moved either upstream or downstreamfrom the promoter. Both enhancers and other upstream promoter elementsbind sequence-specific DNA-binding proteins that mediate their effects.

Vectors for use in accordance with the present invention may beconstructed to include the ocs enhancer element. This element was firstidentified as a 16 bp palindromic enhancer from the octopine synthase(ocs) gene of Agrobacterium (Ellis et al., 1987), and is present in atleast 10 other promoters (Bouchez et al., 1989). The use of an enhancerelement, such as the ocs element and particularly multiple copies of theelement, will act to increase the level of transcription from adjacentpromoters when applied in the context of monocot transformation.

Constructs of the invention will also include the gene of interest alongwith a 3′ end DNA sequence that acts as a signal to terminatetranscription and allow for the polyadenylation of the resultant mRNA.Preferred 3′ elements for plants include those from the nopalinesynthase gene of Agrobacterium tumefaciens (Bevan et al., 1983), theterminator for the T7 transcript from the octopine synthase gene ofAgrobacterium tumefaciens, and the 3′ end of the protease inhibitor I orII genes from potato or tomato. Regulatory elements such as Adh intron 1(Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) orTMV omega element (Gallie, et al., 1989), may further be included wheredesired. Convenient plant termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also, Guerineau et al. (1991);Proudfoot (1991); Sanfacon et al. (1991); Mogen et al. (1990); Munroe etal. (1990); Ballas et al. (1989); Joshi et al. (1987).

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, or direct proteins through theextracellular membrane. In plants, the signal sequence can target thepolypeptide encoded by the polynucleotide to a specific compartmentwithin a plant. Examples of such targets include, but are not limitedto, a vacuole, endoplasmic reticulum, chloroplast, or starch granule. Anexample of a signal sequence includes the maize γ-zein N-terminal signalsequence for targeting to the endoplasmic reticulum and secretion intothe apoplast (Torrent et al., 1997). Another signal sequence is theamino acid sequence SEKDEL for retaining polypeptides in the endoplasmicreticulum (Munro and Pelham, 1987). A polypeptide may also be targetedto the amyloplast by fusion to the waxy amyloplast targeting peptide(Klosgen et al., 1986) or to a starch granule.

For example, it may be useful to target introduced DNA to the nucleus asthis may increase the frequency of transformation. Within the nucleusitself it would be useful to target a gene in order to achieve sitespecific integration. For example, it would be useful to have a geneintroduced through transformation replace an existing gene in the cell.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure. Sequences that are derived from genes that are highlyexpressed in plants will be most preferred.

Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait whichone can select for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by ‘screening’ (e.g., the R-locus trait). Of course, manyexamples of suitable marker genes are known to the art and can beemployed in the practice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; small active enzymes detectable in extracellularsolution (e.g., α-amylase, β-lactamase, phosphinothricinacetyltransferase); and proteins that are inserted or trapped in thecell wall (e.g., proteins that include a leader sequence such as thatfound in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). For example, themaize HPRG (Steifel et al., 1990) molecule is well characterized interms of molecular biology, expression and protein structure. However,any one of a variety of extensins and/or glycine-rich wall proteins(Keller et al., 1989) could be modified by the addition of an antigenicsite to create a screenable marker.

Selectable Markers

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,1985) which codes for kanamycin resistance and can be selected for usingkanamycin, G418, and the like; a bar gene which codes for bialaphosresistance; a gene which encodes an altered EPSP synthase protein(Hinchee et al., 1988) thus conferring glyphosate resistance; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a mutant acetolactatesynthase gene (ALS) which confers resistance to imidazolinone,sulfonylurea or other ALS-inhibiting chemicals (European PatentApplication 154,204, 1985); a methotrexate-resistant DHFR gene (Thilletet al., 1988); a dalapon dehalogenase gene that confers resistance tothe herbicide dalapon; or a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan. Where a mutant EPSP synthasegene is employed, additional benefit may be realized through theincorporation of a suitable chloroplast transit peptide, CTP (EuropeanPatent Application 0,218,571, 1987).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants are the genes that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death. The success in using this selective system in conjunctionwith monocots was particularly surprising because of the majordifficulties which have been reported in transformation of cereals(Potrykus, 1989).

Where one desires to employ a bialaphos resistance gene in the practiceof the invention, a particularly useful gene for this purpose is the baror pat genes obtainable from species of Streptomyces (e.g., ATCC No.21,705). The cloning of the bar gene has been described (Murakami et al.1986; Thompson et al. 1987) as has the use of the bar gene in thecontext of plants other than monocots (De Block et al. 1987; De Block etal. 1989).

Selectable markers for use in prokaryotes include a tetracyclineresistance or an ampillicin resistance gene.

Screenable Markers

Screenable markers that may be employed include, but are not limited to,a β-glucuronidase or uidA gene (GUS) which encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., 1988); a β-lactamase gene(Sutcliffe, 1978), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky et al., 1983) which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an α-amylase gene (Ikuta et al.,1990); a tyrosinase gene (Katz et al., 1983) which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to form the easily detectable compound melanin; aβ-galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), whichallows for bioluminescence detection; or even an aequorin gene (Prasheret al., 1985), which may be employed in calcium-sensitivebioluminescence detection, or a green fluorescent protein gene (Niedz etal., 1995).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. A gene from the R gene complex was appliedto maize transformation, because the expression of this gene intransformed cells does not harm the cells. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline carries dominant alleles for genes encoding the enzymaticintermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1and Bz2), but carries a recessive allele at the R locus, transformationof any cell from that line with R will result in red pigment formation.Exemplary lines include Wisconsin 22 which contains the rg-Stadlerallele and TR112, a K55 derivative which is r-g, b, P1. Alternativelyany genotype of maize can be utilized if the C1 and R alleles areintroduced together. A further screenable marker contemplated for use inthe present invention is firefly luciferase, encoded by the lux gene.The presence of the lux gene in transformed cells may be detected using,for example, X-ray film, scintillation counting, fluorescentspectrophotometry, low-light video cameras, photon counting cameras ormultiwell luminometry. It is also envisioned that this system may bedeveloped for populational screening for bioluminescence, such as ontissue culture plates, or even for whole plant screening.

Transformation

The expression cassette, or a vector construct containing the expressioncassette, may be inserted into a cell. The expression cassette or vectorconstruct may be carried episomally or integrated into the genome of thecell, e.g., derivatives of SV40; bacterial plasmids; phage DNA;baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. However, any vector may be used as long as itis replicable and viable in the host. If the expression cassette isintroduced into a plant cell, a transformed plant cell may be grown intoa transgenic plant. Accordingly, the invention provides transgenicplants and the products of the transgenic plant. Such products mayinclude, but are not limited to, the seeds, fruit, progeny, and productsof the progeny of the transgenic plant.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a cellular host. Transformationof bacteria and many eukaryotic cells may be accomplished through use ofpolyethylene glycol, calcium chloride, viral infection, DEAE dextran,phage infection, electroporation and other methods known in the art.Transformation of fungus may be accomplished according to Gonni et al.(1987). Introduction of the recombinant vector into yeasts can beaccomplished by methods including electroporation, use of spheroplasts,lithium acetate, and the like. Any method capable of introducing DNAinto animal cells can be used: for example, electroporation, calciumphosphate, lipofection and the like.

The expression cassette may be inserted into an insect cell using abaculovirus (See e.g. Baculovirus Expression Vectors, A LaboratoryManual (1992)). For example, the vector into which the recombinant genehas been introduced maybe introduced together with baculovirus into aninsect cell such that a recombinant virus is obtained in the supernatantof the cultured insect cell. Insect cells are then infected with therecombinant virus whereby the protein can be expressed. Thegene-introducing vector used in this method may include e.g. pLV1392,pVL1393, and pBlueBacIII (which all are products of Invitrogen). Thebaculovirus, may be, e.g., Autographa californica nuclear polyhedrosisvirus, which is a virus infecting certain moth insects. The insectcells, may be ovary cells Sf9 and Sf21 from Spodoptera frugiperda andHigh 5 (Invitrogen), which is an ovary cell from Trichoplusia ni, etc.For co-introduction of both the vector having the recombinant gene andthe baculovirus into an insect cell to prepare a recombinant virus, thecalcium phosphate or lipofection methods may be used.

Methods of introducing expression vectors into plant tissue include thedirect infection or co-cultivation of a plant cell with Agrobacteriumtumefaciens (Horsch et al., 1985). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by Gruber et al. (1997). Techniques for transforming plantcells include transformation with DNA employing A. tumefaciens or A.rhizogenes as the transforming agent, electroporation, DNA injection,microprojectile bombardment, particle acceleration, and the like (See,for example, EP 295959 and EP 138341).

It is particularly preferred to use the binary type vectors of Ti and Riplasmids of Agrobacterium spp. Ti-derived vectors transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti etal. 1985; Byrne et al. 1987; Sukhapinda et al. 1987; Lorz et al. 1985;Potrykus 1985; Park et al. 1985; and Hiei et al. 1994). The use of T-DNAto transform plant cells has received extensive study and is amplydescribed (see, e.g., EP 120516; Hoekema 1985; Krauf et al. 1983 and An.et al. 1985).

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs (see EP 295959),techniques of electroporation (Fromm et al. 1986) or high velocityballistic bombardment with metal particles coated with the nucleic acidconstructs (Kline et al. 1987, and U.S. Pat. No. 4,945,050). Oncetransformed, the cells can be regenerated by those skilled in the art.Of particular relevance are the recently described methods to transformforeign genes into commercially important crops, such as rapeseed (DeBlock et al., 1989), sunflower (Everett et al. 1987), soybean (McCabe etal. 1988; Hinchee et al. 1988; Chee et al. 1989; Christou et al. 1989;and EP 301749), rice (Hiei et al., 1994), and corn (Gordon Kamm et al.1990; and Fromm et al., 1990).

Expression vectors containing genomic or synthetic fragments can beintroduced into protoplasts or into intact tissues or isolated cells.Preferably expression vectors are introduced into intact tissue. Generalmethods of culturing plant tissues are provided for example by Maki etal. (1993); and by Phillips et al. (1988).

Preferably, expression vectors are introduced into maize or other planttissues using a direct gene transfer method such asmicroprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using the microprojectile media delivery with the biolisticdevice. See, for example, Tomes et al. (1995).

Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant, i.e., monocotyledonous or dicotyledonous,targeted for transformation. Suitable methods of transforming plantcells include, but are not limited to, microinjection (Crossway et al.1986), electroporation (Riggs et al. 1986), Agrobacterium-mediatedtransformation (De Blaere et al. 1987; and Hinchee et al. 1988), directgene transfer (Paszkowski et al. 1984 cite our patent), and ballisticparticle acceleration using devices available from Agracetus, Inc.,Madison, Wis. and BioRad, Hercules, Calif. (see, for example, Sanford etal., U.S. Pat. No. 4,945,050; and McCabe et al. 1988). Also see,Weissinger et al. 1988; Sanford et al. 1987 (onion); Christou et al.,1988 (soybean); McCabe et al. 1988 (soybean); Datta et al. 1990 (rice);Klein et al. 1988 (maize); Klein et al. 1988 (maize); Klein et al. 1988(maize); Fromm et al. 1990 (maize); and Gordon-Kamm et al. 1990 (maize);Svab et al. 1990 (tobacco chloroplast); Koziel et al. 1993 (maize);Shimamoto et al. 1989 (rice); Christou et al. 1991 (rice); EuropeanPatent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasilet al., 1993 (wheat); Weeks et al. 1993 (wheat); and Methods inMolecular Biology (1998).

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e., co-transformation), and both thesetechniques are suitable for use with the expression cassettes andconstructs of the present invention. Numerous transformation vectors areavailable for plant transformation, and the expression cassettes of thisinvention can be used in conjunction with any such vectors. Theselection of vector will depend upon the preferred transformationtechnique and the target species for transformation.

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19. Bevan (1984). An additional vectoruseful for Agrobacterium-mediated transformation is the binary vectorpCIB 10, which contains a gene encoding kanamycin resistance forselection in plants, T-DNA right and left border sequences andincorporates sequences from the wide host-range plasmid pRK252 allowingit to replicate in both E. coli and Agrobacterium. Its construction isdescribed by Rothstein et al. (1987). Various derivatives of pCIB10 havebeen constructed which incorporate the gene for hygromycin Bphosphotransferase described by Gritz et al. (1983). These derivativesenable selection of transgenic plant cells on hygromycin only (pCIB743),or hygromycin and kanamycin (pCIB715, pCIB717).

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker which may provide resistance to anantibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide(e.g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention.

For certain plant species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra 1982; Bevan et al.1983), the bar gene which confers resistance to the herbicidephosphinothricin (White et al. 1990, Spencer et al. 1990), the hph genewhich confers resistance to the antibiotic hygromycin (Blochinger &Diggelmann), and the dhfr gene, which confers resistance to methotrexate(Bourouis et al. 1983).

One such vector useful for direct gene transfer techniques incombination with selection by the herbicide Basta (or phosphinothricin)is pCIB3064. This vector is based on the plasmid pCIB246, whichcomprises the CaMV 35S promoter in operational fusion to the E. coli GUSgene and the CaMV 35S transcriptional terminator and is described in WO93/07278, herein incorporated by reference. One gene useful forconferring resistance to phosphinothricin is the bar gene fromStreptomyces viridochromogenes (Thompson et al. 1987). This vector issuitable for the cloning of plant expression cassettes containing theirown regulatory signals. An additional transformation vector is pSOG35which utilizes the E. coli gene dihydrofolate reductase (DHFR) as aselectable marker conferring resistance to methotrexate.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a construct ofthe present invention. The term organogenesis means a process by whichshoots and roots are developed sequentially from meristematic centerswhile the term embryogenesis means a process by which shoots and rootsdevelop together in a concerted fashion (not sequentially), whether fromsomatic cells or gametes. The particular tissue chosen will varydepending on the clonal propagation systems available for, and bestsuited to, the particular species being transformed. Exemplary tissuetargets include leaf disks, pollen, embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristems, axillary buds, and root meristems), and inducedmeristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as npt II) can be associated with the expression cassetteto assist in breeding.

The present invention may be used for transformation of any plantspecies, including, but not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, such as canola, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Leguminous plants include beans and peas. Beans include guar, locustbean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean,fava bean, lentils, chickpea, etc. Legumes include, but are not limitedto, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch,adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium,Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean,Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil,lens, e.g., lentil, and false indigo. Preferred forage and turf grassfor use in the methods of the invention include alfalfa, orchard grass,tall fescue, perennial ryegrass, creeping bent grass, and redtop.

Preferably, plants of the present invention are crop plants, forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, oat, rye, millet, tobacco, barley, rice, tomato,potato, squash, melons, legume crops, e.g., pea, bean and soybean, andthe like.

Recombinant Enzyme

For preparation of recombinant phytase, following transformation of asuitable host and growth of the host, a selected promoter may be inducedby appropriate means (e.g., temperature shift or chemical induction) andcells cultured for an additional period to yield recombinant enzyme.Cells are then typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Cells employed in expression of proteins can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents, such methods are well known tothose skilled in the art.

The enzyme can be recovered and purified from recombinant cell culturesby methods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The enzymes of the present invention may be a product of chemicalsynthetic procedures, or produced by recombinant techniques from aeukaryotic host such as a higher plant.

Depending upon the host employed in a recombinant production procedure,the enzyme of the present invention may or may not be covalentlymodified via glycosylation. In eukaryotic cells glycosylation ofsecreted proteins serves to modulate protein folding, conformational andthermostability stability, and resistance to proteolysis. Given aspecific application of phytase use, a glycosylated version of theenzyme may be preferable over a non-glycosylated form. For example, theuse of a glycosylated phytase in animal feed helps protect the enzymefrom thermal denaturation during feed pelleting and from proteolyticinactivation as it passes through the stomach of the animal, helpingdeliver active enzyme to the intestinal tract and site of action. Forfood processing applications where enzyme activity is desired onlyduring processing and not in the final product a non-glycosylated,thermolabile, and proteolytic susceptible phytase is preferred.

Enzymes of the invention may or may not also include an initialmethionine amino acid residue.

The enzyme of this invention may be employed for any purpose in whichsuch enzyme activity is necessary or desired. In a preferred embodiment,the enzyme is employed for catalyzing the hydrolysis of phytate inanimal feed. In another preferred embodiment, the enzyme is employed forcatalyzing the hydrolysis of phytate in food.

Production and Characterization of Stably Transformed Plants

Transformed plant cells are placed in an appropriate selective mediumfor selection of transgenic cells that are then grown to callus. Shootsare grown from callus and plantlets generated from the shoot by growingin rooting medium. The various constructs normally will be joined to amarker for selection in plant cells. Conveniently, the marker may beresistance to a biocide (particularly an antibiotic, such as kanamycin,G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like).The particular marker used will allow for selection of transformed cellsas compared to cells lacking the DNA which has been introduced.Components of DNA constructs, including transcription/expressioncassettes of this invention, may be prepared from sequences which arenative (endogenous) or foreign (exogenous) to the host. By “foreign” itis meant that the sequence is not found in the wild-type host into whichthe construct is introduced. Heterologous constructs will contain atleast one region which is not native to the gene from which thetranscription-initiation-region is derived.

To confirm the presence of the transgenes in transgenic cells andplants, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, in situ hybridizationand nucleic acid-based amplification methods such as PCR or RT-PCR;“biochemical” assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function; plant part assays, such as leaf or root assays; andalso, by analyzing the phenotype of the whole regenerated.

DNA may be isolated from cells or an organism or tissue thereofincluding any plant parts to determine the presence of a particularnucleic acid segment through the use of techniques well known to thoseskilled in the art. Note that intact sequences will not always bepresent, presumably due to rearrangement or deletion of sequences in thecell.

The presence of nucleic acid elements introduced through the methods ofthis invention may be determined by polymerase chain reaction (PCR).Using this technique discreet fragments of nucleic acid are amplifiedand detected by gel electrophoresis. This type of analysis permits oneto determine whether a preselected nucleic acid segment is present in astable transformant, but does not prove integration of the introducedpreselected nucleic acid segment into the host cell genome. In addition,it is not possible using PCR techniques to determine whethertransformants have exogenous genes introduced into different sites inthe genome, i.e., whether transformants are of independent origin. It iscontemplated that using PCR techniques it would be possible to clonefragments of the host genomic DNA adjacent to an introduced particularDNA segment.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced particular DNAsegments in high molecular weight DNA, i.e., confirm that the introducedparticular DNA segment has been integrated into the host cell genome.The technique of Southern hybridization provides information that isobtained using PCR, e.g., the presence of a particular DNA segment, butalso demonstrates integration into the genome and characterizes eachindividual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of a particular DNA segment.

Both PCR and Southern hybridization techniques can be used todemonstrate transmission of a particular DNA segment to progeny. In mostinstances the characteristic Southern hybridization pattern for a giventransformant will segregate in progeny as one or more Mendelian genes(Spencer et al., 1992; Laursen et al., 1994) indicating stableinheritance of the gene. The nonchimeric nature of the callus and theparental transformants (R₀) was suggested by germline transmission andthe identical Southern blot hybridization patterns and intensities ofthe transforming DNA in callus, R₀ plants and R₁ progeny that segregatedfor the transformed gene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced particular DNA segments. Inthis application of PCR it is first necessary to reverse transcribe RNAinto DNA, using enzymes such as reverse transcriptase, and then throughthe use of conventional PCR techniques amplify the DNA. In mostinstances PCR techniques, while useful, will not demonstrate integrityof the RNA product. Further information about the nature of the RNAproduct may be obtained by Northern blotting. This technique willdemonstrate the presence of an RNA species and give information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

Thus, while Southern blotting and PCR may be used to detect theparticular DNA segment in question, they do not provide information asto whether the particular DNA segment is being expressed. Expression maybe evaluated by specifically identifying the protein products of theintroduced particular DNA segments or evaluating the phenotypic changesbrought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures may also be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to beanalyzed.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant

Phytase Compositions

Generally, phytase compositions are liquid or dry.

Liquid compositions need not contain anything more than the phytaseenzyme, preferably in a highly purified form. However, a stabilizer suchas glycerol, sorbitol or mono propylen glycol may be added. The liquidcomposition may also comprise other additives, such as salts, sugars,preservatives, pH-adjusting agents, proteins, and phytate (a phytasesubstrate). Typical liquid compositions are aqueous or oil-basedslurries. The liquid compositions may be added to a food or feed beforeor after an optional pelleting thereof.

Dry compositions may be freeze-dried or spray dried compositions, inwhich case the composition need not contain anything more than theenzyme in a dry form Dry compositions may be granulates which mayreadily be mixed with, e.g., food or feed components, or morepreferably, form a component of a pre-mix. The particle size of theenzyme granulates preferably is compatible with that of the othercomponents of the mixture. This provides a safe and convenient means ofincorporating enzymes into, e.g., processed food or animal feed.

For example, a stable phytase enzyme formulation can be prepared byfreezing a mixture of liquid enzyme solution with a bulking agent suchas ground soybean meal, and then lyophilizing the mixture. The reductionin moisture and the binding interactions of the phytase with the bulkingagent protect the enzyme from external environmental factors such as thetemperature extremes experienced during compound feed manufacture. Dryformulations can further enhance stability by minimizing the activity ofpotential proteolytic enzymes that may be present as by-products in theliquid fermentation mixture used to manufacture the target enzyme. Theresulting dry enzyme-soy flour mixture can withstand high extremes oftemperature. For example, after 120 minutes of heating at 96° C., thedry enzyme formulation retained 97.8% of its original enzymaticactivity. The formulated enzyme mixture can be used as a feed supplementfor use in poultry and swine production. For instance, addition of 500enzyme units of a thermotolerant phytase of the invention to 1 kg of astandard corn-soy poultry diet will allow a reduction in the levels ofinorganic phosphate supplementation currently used in animal nutrition,i.e., from 0.45% to 0.225%. Chickens raised on a 0.225% phosphate dietsupplemented with the formulated phytase will perform as well aschickens fed a standard diet containing 0.45% phosphate. Moreover, areduction in phosphate supplementation results in decreased levels ofphosphate pollution, which in turn significantly lessens theenvironmental impact of intensive commercial animal production.

Potential drawbacks with using small particle size dry formulations aretheir dust forming tendencies and the high local concentration of thetarget enzyme on the surface of the particles. Dust particlesimpregnated with enzyme protein may pose an immunological concern, whilelocalization of enzyme predominately on the surface of the smallparticles may affect stability, particularly during prolonged period ofstorage and during feed manufacture.

Thus, further provided by the invention is a non-manufactured method offormulation, which comprises producing and delivering the target enzymeof the invention in grain such as maize, wheat, or soy. The intact grainprotects the target enzyme from external environmental factors andminimizes the production of dust. This method of enzyme delivery addssignificant savings to a formulation cost, particularly when compared tothe cost of low-dust granulate formulations currently used commercially.The grain-containing enzyme may be added to animal feed in the form ofcracked seed, ground seed, or in a more refined form. Alternatively, aprotein extract may be made from seed, and that extract can be furtherprocessed into either a stabilized liquid or into a dry state bylyophilization or spray drying. For example, enzymatically activephytase can be produced in maize at a level of 1,000,000 units per kg ofseed, and active enzyme is recoverable from the seed by aqueousextraction.

Agglomeration granulates are prepared using agglomeration techniques ina high shear mixer during which a filler material and the enzyme areco-agglomerated to form granules. Absorption granulates are prepared byhaving cores of a carrier material to absorb/be coated by the enzyme.

Typical filler materials are salts such as disodium sulphate. Otherfillers include kaolin, talc, magnesium aluminium silicate and cellulosefibres. Optionally, binders such as dextrins are also included inagglomeration granulates.

Typical carrier materials include starch, e.g., in the form of cassaya,corn, potato, rice and wheat. Salts may also be used.

Optionally, the granulates are coated with a coating mixture. Such amixture comprises coating agents, preferably hydrophobic coating agents,such as hydrogenated palm oil and beef tallow, and if desired, otheradditives such as calcium carbonate or kaolin.

Additionally, phytase compositions may contain other substituents suchas coloring agents, aroma compounds, stabilizers, vitamins, minerals,other feed or food enhancing enzymes etc. This is so in particular forthe so-called pre-mixes.

A “food or feed additive” is an essentially pure compound or a multicomponent composition intended for or suitable for being added to foodor feed. In particular it is a substance that by its intended use isbecoming a component of a food or feed product or affects anycharacteristics of a food or feed product. Thus, a phytase additive isunderstood to mean a phytase which is not a natural constituent of themain feed or food substances or is not present at its naturalconcentration therein, e.g., the phytase is added to the feed separatelyfrom the feed substances, alone or in combination with other feedadditives, or the phytase is an integral part of one of the feedsubstances but has been produced therein by recombinant DNA technology.A typical additive usually comprises one or more compounds such asvitamins, minerals or feed enhancing enzymes and suitable carriersand/or excipients.

A ready for use phytase additive is herein defined as an additive thatis not produced in situ in animal feed or in processed food. A ready foruse phytase additive may be fed to humans or animals directly or,preferably, directly after mixing with other feed or food constituents.For example, a feed additive according to this aspect of the presentinvention is combined with other feed components to produce feed. Suchother feed components include one or more other (preferablythermostable) enzyme supplements, vitamin feed additives, mineral feedadditives and amino acid feed additives. The resulting (combined) feedadditive including possibly several different types of compounds canthen be mixed in an appropriate amount with the other feed componentssuch as cereal and protein supplements to form an animal feed.Processing of these components into an animal feed can be performedusing any of the currently used processing apparatuses such as adouble-pelleting machine, a steam pelleter, an expander or an extruder.

Similarly, a food additive according to this aspect of the presentinvention is combined with other food components to produce processedfood products. Such other food components include one or more other(preferably thermostable) enzyme supplements, vitamin food additives andmineral food additives. The resulting (combined) food additive,including possibly several different types of compounds can then bemixed in an appropriate amount with the other food components such ascereal and plant proteins to form a processed food product. Processingof these components into a processed food product can be performed usingany of the currently used processing apparatuses.

In a preferred embodiment, the phytase compositions of the inventionadditionally comprises an effective amount of one or more feed or foodenhancing enzymes, in particular feed or food enhancing enzymes selectedfrom the group consisting of α-galactosidases, β-galactosidases, inparticular lactases, other phytases, β-glucanases, in particularendo-β-1,4-glucanases and endo-β-1,3(4)-glucanases, cellulases,xylosidases, galactanases, in particular arabinogalactanendo-1,4-β-galactosidases and arabinogalactan endo-1,3-β-galactosidases,endoglucanases, in particular endo-1,2-β-glucanase,endo-1,3-α-glucanase, and endo-1,3-β-glucanase, pectin degradingenzymes, in particular pectinases, pectinesterases, pectin lyases,polygalacturonases, arabinanases, rhamnogalacturonases,rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α-rhamnosidase,pectate lyases, and α-galacturonisidases, mannanases, β-mannosidases,mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phospholipasesand cutinases.

The animal feed additive of the invention is supplemented to the animalbefore or simultaneously with the diet. Preferably, the animal feedadditive of the invention is supplemented to the animal simultaneouslywith the diet.

An effective amount of phytase in food or feed is from about 10 to20,000 FTU/kg; preferably from about 10 to 15,000 FTU/kg, morepreferably from about 10 to 10,000 FTU/kg, in particular from about 100to 5,000 FTU/kg, especially from about 100 to about 2,000 FTU/kg feed orfood.

Also within the scope of this invention is the use of phytase forprocessing and manufacturing human foods and animal feeds. Grains andflours destined for human foods can be enzymatically treated withphytase to reduce the phytin content of the material. The reduced levelsof phytin enhance the quality of the food by increasing the nutrientavailability of essential minerals such as iron, calcium, and zinc. Inaddition to increasing the nutritional quality of food, phytase usedduring food processing can improve the overall efficiency of the foodproduction method. For example, addition of phytase to white soybeanflakes during soy protein isolate manufacturing can significantlyincrease the yield and quality of extractable protein. During foodmanufacture the phytase is active during manufacture and processingonly, and is not active in the final food product. This aspect isrelevant for instance in dough making and baking. Similarly, animal feedgrain, such as toasted soybean meal or canola meal, may be pre-processedwith phytase prior to compound feed manufacture. Removal of theanti-nutritive factors in animal feed components prior to compound feedmanufacture produces a nutritionally higher quality and more valuableanimal feed ingredient. In this processing method the phytase is activeduring feed manufacturing, and may or may not be active in the digestivetract of the animal upon ingestion of the treated feed.

In addition to using phytase as a food processing aid, the scope of thisinvention encompasses the use of phytase as a human supplementaldigestive aid. Phytase in tablet form can be ingested at the time offood consumption to deliver active enzyme to the gastrointestinal tractof the recipient. Nutritional gains for the consumer would beexperienced in vivo and may be taken with foods that cannot be treatedwith a phytase during food processing.

Also within the scope of the invention is the use of a phytase of theinvention during the preparation of food or feed preparations oradditives, i.e., the phytase is active during the manufacture only andis not active in the final food or feed product. This aspect isparticularly relevant, for instance, in dough making and baking and theproduction of other ready-to-eat cereal based products.

Another possibility for the exogenous addition of phytase to animal feedand processed food is to add phytase-containing transgenic plantmaterial to the feed, preferably processed transgenic seed, in which thephytase has been synthesized through heterologous gene expression. Theparts of the plants which express the heterologous phytase, e.g., theseed of the transgenic plants or other plant materials such as roots,stems, leaves, wood, flowers, bark, and/or fruit may be included inanimal feed, either as such or after further processing. In acereal-based feed or food, the cereal is preferably wheat, barley,maize, sorghum, rye, oats, triticale or rice. The phytase may also beused advantageously in monogastrics as well as in polygastrics,especially young calves. Diets for fish and crustaceans may also besupplemented with phytase to further improve feed conversion ratio andreduce the level of excreted phosphorus for intensive productionsystems. The feed according to the present invention may also beprovided to animals such as poultry, e.g., turkeys, geese, ducks, aswell as swine, equine, bovine, ovine, caprine, canine and feline, aswell as fish and crustaceans. It is however particularly preferred thatthe feed is provided to pigs or to poultry, including, but not limitedto, broiler chickens, hens, in particular laying hens, turkeys andducks.

Feed Compositions and Methods of Use

The phytases (formulated as described above) of the current inventionmay be combined with other ingredients to result in novel feedcompositions with particular advantages.

For instance, it is preferable that intensive animal productionoperations limit the phosphate pollution that is contained in the fecesof the animals that are produced. The amount of phosphate present in thediet and the availability of the phosphate in the diet to the animal arethe primary factors influencing the excreted phosphate present in thefeces of the animal. Currently, the availability of the plant, orgrain-derived phosphate, present in soybean meal, corn grain (and otherfeedstuffs) is low as the phosphate is primarily in the form of phyticacid. In order to maximize the growth efficiencies of the animalsinorganic phosphate is added to feed resulting in a feed compositionthat contains adequate levels of available phosphate. However, thesefeed formulations contain too much total phosphate and result inphosphate pollution.

Although commercially available phytases at present result in higherphosphate availability they are recommended to be used with high levelsof added inorganic phosphate. The phytases of the present invention areso active that they can be used to create novel animal feed formulationsthat have a) significantly reduced levels of inorganic phosphate, and b)allow superior feed conversion efficiency and improved weight gainrelative to normal diets. At present, commercially available phytaseswill not allow animals to be efficiently produced on a feed thatcontains no added inorganic phosphorus

Specifically, the animal feed of the invention comprises the combinationof a phytase of the present invention in combination with animal feedingredients to form a feed that has substantially lowered inorganicphosphorus levels. In a preferred embodiment, the feed compositions ofthe invention comprises typical feed ingredients, micronutrients,vitamins, etc. and an effective amount of thermostable phytase andinorganic phosphate where the amounts of the phytase and phosphorus arefrom about between the levels of 50-20,000 units of phytase per kg offeed and less than 0.45% inorganic phosphorus; preferably between thelevels of 100-10,000 units of phytase per kg of feed and less than0.225% inorganic phosphorus; in particular between the levels of150-10,000 units of phytase per kg of feed and less than 0.15% inorganicphosphorus, or especially between the levels of 250-20,000 units ofphytase per kg of feed and no exogenously added inorganic phosphorus.

Also, within the scope of the invention are methods of improving weightgains, and feed conversions ratios (FCR) associated with production offarm animals. A phytase of the present invention allows improved weightgains and FCR especially when used in combination with diets that arelow in inorganic phosphate. Specifically the method of the presentinvention to improve the FCR, or weight gain of a low inorganicphosphate diet by feeding a diet to an animal comprising a phytase ofthe present invention and a level of inorganic phosphate at or below thelevel of 0.45%. Preferably, the method comprises feeding a dietcontaining the phytase and less than 0.225% inorganic phosphate, or mostpreferably the method comprises feeding a diet containing the phytaseand no added inorganic phosphorus.

The animal feed of the present invention can be used on monogastric orpolygastric animals. The animal feed of the present invention can befeed for poultry, or swine, or calves, or companion animals such as dogsor cats or horsed. Examples of such feed and the use of the feed areprovided in Example 3.

The present invention also provides for a method of animal husbandrythat results in a significantly reduced environmental phosphate load.The method comprises feeding entire flocks or herds of farm animals afeed composition containing a phytase of the present invention and areduced amount of inorganic phosphorus (less than 0.45%). Morepreferably the method comprises feeding entire flocks or herds of farmanimals a feed composition containing a phytase of the present inventionand a significantly reduced amount of inorganic phosphorus (less than0.225%), or most preferably the method comprising feeding entire flocksor herds of farm animals a feed composition containing a phytase of thepresent invention and no inorganic phosphorus. This method will allowhigh densities of animals to be maintained while minimizing theenvironmental release of phosphate from the farming operation.

Methods Useful for the Invention

I. Expression Cassettes Useful for the Invention

Coding sequences intended for expression in transgenic plants are firstassembled in expression cassettes 3′ to a suitable promoter expressiblein plants. The expression cassettes may also comprise any furthersequences required or selected for the expression of the transgene. Suchsequences include, but are not restricted to, transcription terminators,extraneous sequences to enhance expression such as introns, vitalsequences, and sequences intended for the targeting of the gene productto specific organelles and cell compartments. These expression cassettescan then be transferred to the plant transformation vectors describedherein.

The following is a description of various components of typicalexpression cassettes.

A. Promoters

Selection of the promoter to be used in expression cassettes willdetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, mesophyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and selection should reflect the desired locationof accumulation of the gene product. Alternatively, the selectedpromoter may drive expression of the gene under various inducingconditions. Promoters vary in their strength, i.e., ability to promotetranscription. Depending upon the host cell system utilized, any one ofa number of suitable promoters can be used, including the gene's nativepromoter. The following are non-limiting examples of promoters that maybe used in the expression cassettes employed in the present invention.

Constitutive Promoters

a. Ubiquitin Promoters

Ubiquitin is a gene product known to accumulate in many cell types andits promoter has been cloned from several species for use in transgenicplants (e.g. sunflower—Binet et al. Plant Science 79: 87-94 (1991);maize—Christensen et al. Plant Molec. Biol. 12: 619-632 (1989); andArabidopsis—Norris et al., Plant Mol. Biol. 21:895-906 (1993)). Themaize ubiquitin promoter has been developed in transgenic monocotsystems and its sequence and vectors constructed for monocottransformation are disclosed in the patent publication EP 0 342 926 (toLubrizol), which is herein incorporated by reference. Taylor et al.(Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) thatcomprises the maize ubiquitin promoter and first intron and its highactivity in cell suspensions of numerous monocotyledons when introducedvia microprojectile bombardment. The Arabidopsis ubiquitin promoter isideal for use with the nucleotide sequences of the present invention.The ubiquitin promoter is suitable for gene expression in transgenicplants, both monocotyledons and dicotyledons. Suitable vectors includederivatives of pAHC25 or any of the transformation vectors described inthis application. The vectors may be modified by introducing of theappropriate ubiquitin promoter and/or intron sequences.

b. The CaMV 35S Promoter

Construction of the plasmid pCGN1761 is described in published patentapplication EP 0 392 225 (published Sep. 25, 1991; Ciba Geigy; Example23), which is hereby incorporated by reference. The plasmid contains the“double” CaMV 35S promoter and the tml transcriptional terminator with aunique EcoRI site between the promoter and the terminator and has apUC-type backbone. A derivative of pCGN1761 is constructed which has amodified polylinker which includes NotI and XhoI sites in addition tothe existing EcoRI site. This derivative, designated pCGN1761ENX and isuseful for the cloning of cDNA sequences or coding sequences (includingmicrobial ORF sequences) within its polylinker for the purpose of theirexpression under the control of the 35S promoter in transgenic plants.The entire 35S promoter-coding sequence-tml terminator cassette of sucha construction can be excised by HindIII, SphI, SalI, and XbaI sites 5′to the promoter and XbaI, BamHI and BglI sites 3′ to the terminator fortransfer to transformation vectors such as those described below.Furthermore, the double 35S promoter fragment can be removed by 5′excision with HindIII, SphI, SalI, XbaI, or PstI, and 3′ excision withany of the polylinker restriction sites (EcoRI, NotI or XhoI) forreplacement with another promoter. If desired, modifications around thecloning sites can be made by the introduction of sequences that mayenhance translation. This is particularly useful when overexpression isdesired. For example, pCGN1761ENX may be modified by optimization of thetranslational initiation site as described in Example 37 of U.S. Pat.No. 5,639,949 (issued Jun. 17, 1997 to Ciba Geigy), incorporated hereinby reference.

c. The Actin Promoter:

Several isoforms of actin are known to be expressed in most cell typesand consequently the actin promoter is a good choice for a constitutivepromoter. In particular, the promoter from the rice ActI gene has beencloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)).A 1.3 kb fragment of the promoter was found to contain all theregulatory elements required for expression in rice protoplasts.Furthermore, numerous expression vectors based on the ActI promoter havebeen constructed specifically for use in monocotyledons (McElroy et al.Mol. Gen. Genet. 231: 150-160 (1991)). These incorporate the ActI-intron1, AdhI 5′ flanking sequence and AdhI-intron I (from the maize alcoholdehydrogenase gene) and sequence from the CaMV 35S promoter. Vectorsshowing highest expression were fusions of 35S and ActI intron or theActI 5′ flanking sequence and the ActI intron. Optimization of sequencesaround the initiating ATG (of the GUS reporter gene) also enhancedexpression. The promoter expression cassettes described by McElroy etal. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified forgene expression and are particularly suitable for use inmonocotyledonous hosts. For example, promoter-containing fragments maybe removed from the McElroy constructions and used to replace the double35S promoter in pCGN1761ENX, which is then available for the insertionof specific gene sequences. The fusion genes thus constructed may thenbe transferred to appropriate transformation vectors. In a separatereport, the rice ActI promoter with its first intron has also been foundto direct high expression in cultured barley cells (Chibbar et al. PlantCell Rep. 12: 506-509 (1993)).

Inducible Expression

a. PR-1 Promoters:

The double 35S promoter in pCGN1761ENX may be replaced with any otherpromoter of choice that will result in suitably high expression levels.By way of example, one of the chemically regulatable promoters describedin U.S. Pat. No. 5,614,395 (issued Mar. 25, 1997 to Ciba Geigy), such asthe tobacco PR-1a promoter, may replace the double 35S promoter.Alternately, the Arabidopsis PR-1 promoter described in Lebel et al.,Plant J. 16:223-233 (1998) may be used. The promoter of choice ispreferably excised from its source by restriction enzymes, but canalternatively be PCR-amplified using primers that carry appropriateterminal restriction sites. Should PCR-amplification be undertaken, thenthe promoter should be re-sequenced to check for amplification errorsafter the cloning of the amplified promoter in the target vector. Thechemically/pathogen regulatable tobacco PR-1a promoter is cleaved fromplasmid pCIB1004 (for construction, see example 21 of EP 0 332 104(published Mar. 20, 1991; Ciba Geigy), which is hereby incorporated byreference) and transferred to plasmid pCGN1761ENX (Uknes et al., PlantCell 4: 645-656 (1992)). The plasmid pCIB1004 is cleaved with NcoI andthe resultant 3′ overhang of the linearized fragment is rendered bluntby treatment with T4 DNA polymerase. The fragment is then cleaved withHindIII and the resultant PR-1a promoter-containing fragment is gelpurified and cloned into pCGN1761ENX from which the double 35S promoterhas been removed. This is done by cleavage with XhoI and blunting withT4 polymerase, followed by cleavage with HindIII and isolation of thelarger vector-terminator containing fragment into which the pCIB1004promoter fragment is cloned. This generates a pCGN1761ENX derivativewith the PR-1a promoter and the tml terminator and an interveningpolylinker with unique EcoRI and NotI sites. The selected codingsequence can be inserted into this vector, and the fusion products (i.e.promoter-gene-terminator) can subsequently be transferred to anyselected transformation vector, including those described infra. Variouschemical regulators may be employed to induce expression of the selectedcoding sequence in the plants transformed according to the presentinvention, including the benzothiadiazole, isonicotinic acid, andsalicylic acid compounds disclosed in U.S. Pat. Nos. 5,523,311 and5,614,395.

b. Ethanol-Inducible Promoters

A promoter inducible by certain alcohols or ketones, such as ethanol,may also be used to confer inducible expression of a coding sequence ofthe present invention. Such a promoter is, for example, the alcA genepromoter from Aspergillus nidulans (Caddick et al. (1998) Nat.Biotechnol 16:177-180). In A. nidulans, the alcA gene encodes alcoholdehydrogenase I, the expression of which is regulated by the AlcRtranscription factors in presence of the chemical inducer. For thepurposes of the present invention, the CAT coding sequences in plasmidpalcA:CAT comprising a alcA gene promoter sequence fused to a minimal35S promoter (Caddick et al. (1998) Nat. Biotechnol 16:177-180) arereplaced by a coding sequence of the present invention to form anexpression cassette having the coding sequence under the control of thealcA gene promoter. This is carried out using methods well known in theart.

c. Glucocorticoid-Inducible Promoter:

Induction of expression of a nucleic acid sequence of the presentinvention using systems based on steroid hormones is also contemplated.For example, a glucocorticoid-mediated induction system is used (Aoyamaand Chua (1997) The Plant Journal 11: 605-612) and gene expression isinduced by application of a glucocorticoid, such as a syntheticglucocorticoid, preferably dexamethasone, preferably at a concentrationranging from 0.1 mM to 1 mM, more preferably from 10 mM to 100 mM. Forthe purposes of the present invention, the luciferase gene sequences arereplaced by a nucleic acid sequence of the invention to form anexpression cassette having a nucleic acid sequence of the inventionunder the control of six copies of the GAL4 upstream activatingsequences fused to the 35S minimal promoter. This is carried out usingmethods well known in the art. The trans-acting factor comprises theGAL4 DNA-binding domain (Keegan et al. (1986) Science 231: 699-704)fused to the transactivating domain of the herpes viral protein VP16(Triezenberg et al. (1988) Genes Devel. 2: 718-729) fused to thehormone-binding domain of the rat glucocorticoid receptor (Picard et al.(1988) Cell 54: 1073-1080). The expression of the fusion protein iscontrolled by any promoter suitable for expression in plants known inthe art or described here. This expression cassette is also comprised inthe plant comprising a nucleic acid sequence of the invention fused tothe 6×GAL4/minimal promoter. Thus, tissue- or organ-specificity of thefusion protein is achieved leading to inducible tissue- ororgan-specificity of the expression cassettes of the present invention.

d. Wound-Inducible Promoters:

Wound-inducible promoters may also be suitable for gene expression.Numerous such promoters have been described (e.g. Xu et al. Plant Molec.Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989),Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al.Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201(1993)) and all are suitable for use with the instant invention.Logemann et al. describe the 5′ upstream sequences of the dicotyledonouspotato wunI gene. Xu et al. show that a wound-inducible promoter fromthe dicotyledon potato (pin2) is active in the monocotyledon rice.Further, Rohrmeier & Lehle describe the cloning of the maize WipI cDNAwhich is wound induced and which can be used to isolate the cognatepromoter using standard techniques. Similar, Firek et al. and Warner etal. have described a wound-induced gene from the monocotyledon Asparagusofficinalis, which is expressed at local wound and pathogen invasionsites. Using cloning techniques well known in the art, these promoterscan be transferred to suitable vectors, fused to the genes pertaining tothis invention, and used to express these genes at the sites of plantwounding.

3. Tissue-Specific Expression

a. Root Specific Expression:

Another pattern of gene expression is root expression. A suitable rootpromoter for the constructs and methods of the present invention is thepromoter of the maize metallothionein-like (MTL) gene described by deFramond (FEBS 290: 103-106 (1991)) and also in U.S. Pat. No. 5,466,785(issued Nov. 14, 1995 to Ciba Geigy), incorporated herein by reference.This “MTL” promoter is transferred to a suitable vector such aspCGN1761ENX for the insertion of a selected gene and subsequent transferof the entire promoter-gene-terminator cassette to a transformationvector of interest.

b. Pith-Preferred Expression:

Patent Application WO 93/07278 (published Apr. 15, 1993; Ciba Geigy),which is herein incorporated by reference, describes the isolation ofthe maize trpA gene, which is preferentially expressed in pith cells.The gene sequence and promoter extending up to −1726 bp from the startof transcription are presented. Using standard molecular biologicaltechniques, this promoter, or parts thereof, can be transferred to avector such as pCGN1761 where it can replace the 35S promoter and beused to drive the expression of a foreign gene in a pith-preferredmanner. In fact, fragments containing the pith-preferred promoter orparts thereof can be transferred to any vector and modified for utilityin transgenic plants.

c. Leaf-Specific Expression:

A maize gene encoding phosphoenol carboxylase (PEPC) has been describedby Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Usingstandard molecular biological techniques the promoter for this gene canbe used to drive the expression of any gene in a leaf-specific manner intransgenic plants.

d. Pollen-Specific Expression:

WO 93/07278 (published Apr. 15, 1993; Ciba Geigy) describes theisolation of the maize calcium-dependent protein kinase (CDPK) genewhich is expressed in pollen cells. The gene sequence and promoterextend up to 1400 bp from the start of transcription. Using standardmolecular biological techniques, this promoter or parts thereof, can betransferred to a vector such as pCGN1761 where it can replace the 35Spromoter and be used to drive the expression of a nucleic acid sequenceof the invention in a pollen-specific manner.

B. Transcriptional Terminators

A variety of transcriptional terminators are available for use in theexpression cassettes of the present invention. These are responsible forthe termination of transcription beyond the transgene and correct mRNApolyadenylation. Suitable transcriptional terminators are those that areknown to function in plants and include, but are not limited to, theCaMV 35S terminator, the tml terminator, the nopaline synthaseterminator and the pea rbcS E9 terminator. These can be used in bothmonocotyledons and dicotyledons. In addition, a gene's nativetranscription terminator may be used.

C. Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize AdhI gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronze1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).Other leader sequences known in the art include but are not limited to:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein. O., Fuerst, T. R., and Moss, B. PNAS USA86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chainbinding protein (BiP) leader, (Macejak, D. G., and Sarnow. P., Nature353: 90-94 (1991); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallie,D. R. et al., Molecular Biology of RNA, pages 237-256 (1989); and MaizeChlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology81:382-385 (1991). See also, Della-Cioppa et al., Plant Physiology84:965-968 (1987).

D. GC/AT Content

It is known in the art that the optimization of protein expression inplants may be enhanced by optimizing the coding regions of genes to thecodon preference of the host. Accordingly, the preferred codon usage inplants differs from the preferred codon usage in certain microorganisms.Comparison of the usage of codons within a cloned microbial ORF to usagein plant genes (and in particular genes from the target plant) willenable an identification of the codons within the ORF which shouldpreferably be changed. Typically plant evolution has tended towards astrong preference of the nucleotides C and G in the third base positionof monocotyledons, whereas dicotyledons often use the nucleotides A or Tat this position. By modifying a gene to incorporate preferred codonusage for a particular target transgenic species, many of the problemsdescribed below for GC/AT content and illegitimate splicing will beovercome.

Plant genes typically have a GC content of more than 35%. ORF sequenceswhich are rich in A and T nucleotides can cause several problems inplants. Firstly, motifs of ATTTA are believed to cause destabilizationof messages and are found at the 3′ end of many short-lived mRNAs.Secondly, the occurrence of polyadenylation signals such as AATAAA atinappropriate positions within the message is believed to causepremature truncation of transcription. In addition, monocotyledons mayrecognize AT-rich sequences as introns and may identify flanking splicesites (see below).

E. Sequences Adjacent to the Initiating Methionine

Plants differ from microorganisms in that their messages do not possessa defined ribosome binding site. Rather, it is believed that ribosomesattach to the 5′ end of the message and scan for the first available ATGat which to start translation. Nevertheless, it is believed that thereis a preference for certain nucleotides adjacent to the ATG and thatexpression of microbial genes can be achieved by the inclusion of aeukaryotic consensus translation initiator at the ATG. Clontech(1993/1994 catalog, page 210, incorporated herein by reference) havesuggested one sequence as a consensus translation initiator for theexpression of the E. coli uidA gene in plants. Further, Joshi (NAR 15:6643-6653 (1987), incorporated herein by reference) has compared manyplant sequences adjacent to the ATG and suggests another consensussequence. In situations where difficulties are encountered in theexpression of microbial ORFs in plants, inclusion of one of thesesequences at the initiating ATG may improve translation. In such casesthe last three nucleotides of the consensus may not be appropriate forinclusion in the modified sequence due to their modification of thesecond AA residue. Preferred sequences adjacent to the initiatingmethionine may differ between different plant species. A survey of 14maize genes located in the GenBank database provided the followingresults: Position Before the Initiating ATG in 14 Maize Genes: −10 −9 −8−7 −6 −5 −4 −3 −2 −1 C3 8 4 6 2 5 6 0 10 7 T3 0 3 4 3 2 1 1 1 0 A2 3 1 43 2 3 7 2 3 G6 3 6 0 6 5 4 6 1 5This analysis can be done for the desired plant species into which thenucleotide sequence is being incorporated, and the sequence adjacent tothe ATG modified to incorporate the preferred nucleotides.

Genes cloned from non-plant sources and not optimized for expression inplants may also contain motifs which may be recognized in plants as 5′or 3′ splice sites, and be cleaved, thus generating truncated or deletedmessages. These sites can be removed using techniques well known in theart.

Techniques for modifying of coding sequences and adjacent sequences arewell known in the art. In cases where the initial expression of amicrobial ORF is low and it is deemed appropriate to make alterations tothe sequence as described above, then the construction of syntheticgenes can be accomplished according to methods well known in the art.These are, for example, described in the published patent disclosures EP0 385 962 (published in Sep. 5, 1990 to Monsanto), EP 0 359 472 (issuedDec. 27, 1995 to Lubrizol) and WO 93/07278 (published Apr. 15, 1993 toCiba-Geigy), all of which are incorporated herein by reference. In mostcases it is preferable to assay the expression of gene constructionsusing transient assay protocols (which are well known in the art) priorto transferring to transgenic plants.

II. Plant Transformation Vectors and Selectable Markers

Numerous transformation vectors available for plant transformation areknown to those of ordinary skill in the plant transformation arts, andthe genes pertinent to this invention can be used in conjunction withany such vectors. The selection of vector will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptII gene, which confers resistance tokanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268(1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, whichconfers resistance to the herbicide phosphinothricin (White et al.,Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79:625-631 (1990)), the hph gene, which confers resistance to theantibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4:2929-2931), and the dhfr gene, which confers resistance to methotrexate(Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, whichconfers resistance to glyphosate (U.S. Pat. Nos. 4,940,835 and5,188,642, issued Jul. 10, 1990 and Feb. 23, 1993, respectively both toMonsanto), and the mannose-6-phosphate isomerase gene (also referred toherein as the phosphomannose isomerase gene), which provides the abilityto metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629, issuedJun. 16, 1998 and Nov. 30, 1999, respectively both to Novartis).

Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)).Below, the construction of two typical vectors suitable forAgrobacterium transformation is described.

pCIB200 and pCIB2001

The binary vectors pcIB200 and pCIB2001 are used for the construction ofrecombinant vectors for use with Agrobacterium and are constructed inthe following manner. pTJS75kan is created by NarI digestion of pTJS75(Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985)) allowingexcision of the tetracycline-resistance gene, followed by insertion ofan AccI fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride etal., Plant Molecular Biology 14: 266-276 (1990)). XhoI linkers areligated to the EcoRV fragment of PCIB7 which contains the left and rightT-DNA borders, a plant selectable nos/nptII chimeric gene and the pUCpolylinker (Rothstein et al., Gene 53: 153-161 (1987)), and theXhoI-digested fragment are cloned into SalI-digested pTJS75kan to createpCIB200 (see also EP 0 332 104, example 19; published Mar. 20, 1991;Ciba Geigy). pCIB200 contains the following unique polylinkerrestriction sites: EcoRI, SstI, KpnI, BglII, XbaI, and SalI. pCIB2001 isa derivative of pCIB200 created by the insertion into the polylinker ofadditional restriction sites. Unique restriction sites in the polylinkerof pCIB2001 are EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI, BclI, AvrII,ApaI, HpaI, and StuI. pCIB2001, in addition to containing these uniquerestriction sites also has plant and bacterial kanamycin selection, leftand right T-DNA borders for Agrobacterium-mediated transformation, theRK2-derived trfA function for mobilization between E. coli and otherhosts, and the OriT and OriV functions also from RK2. The pCIB2001polylinker is suitable for the cloning of plant expression cassettescontaining their own regulatory signals.

pCIB10 and Hygromycin Selection Derivatives Thereof:

The binary vector pCIB10 contains a gene encoding kanamycin resistancefor selection in plants and T-DNA right and left border sequences andincorporates sequences from the wide host-range plasmid pRK252 allowingit to replicate in both E. coli and Agrobacterium. Its construction isdescribed by Rothstein et al. (Gene 53: 153-161 (1987)). Variousderivatives of pCIB10 are constructed which incorporate the gene forhygromycin B phosphotransferase described by Gritz et al. (Gene 25:179-188 (1983)). These derivatives enable selection of transgenic plantcells on hygromycin only (pCIB743), or hygromycin and kanamycin(pCIB715, pCIB717).

Vectors Suitable for Non-Agrobacterium Transformation

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques that do not rely on Agrobacteriuminclude transformation via particle bombardment, protoplast uptake (e.g.PEG and electroporation) and microinjection. The choice of vectordepends largely on the preferred selection for the species beingtransformed. Below, the construction of typical vectors suitable fornon-Agrobacterium transformation is described.

pCIB3064:

pCIB3064 is a pUC-derived vector suitable for direct gene transfertechniques in combination with selection by the herbicide basta (orphosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoterin operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in the PCT publishedapplication WO 93/07278 (published Apr. 15, 1993; Ciba Geigy). The 35Spromoter of this vector contains two ATG sequences 5′ of the start site.These sites are mutated using standard PCR techniques in such a way asto remove the ATGs and generate the restriction sites SspI and PvuII.The new restriction sites are 96 and 37 bp away from the unique SalIsite and 101 and 42 bp away from the actual start site. The resultantderivative of pCIB246 is designated pCIB3025. The GUS gene is thenexcised from pCIB3025 by digestion with Sail and SacI, the terminirendered blunt and religated to generate plasmid pCIB3060. The plasmidpJIT82 may be obtained from the John Innes Centre, Norwich and the a 400bp SmaI fragment containing the bar gene from Streptomycesviridochromogenes is excised and inserted into the HpaI site of pCIB3060(Thompson et al. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064,which comprises the bar gene under the control of the CaMV 35S promoterand terminator for herbicide selection, a gene for ampicillin resistance(for selection in E. coli) and a polylinker with the unique sites SphI,PstI, HindIII, and BamHI. This vector is suitable for the cloning ofplant expression cassettes containing their own regulatory signals.

pSOG19 and pSOG35:

The plasmid pSOG35 is a transformation vector that utilizes the E. coligene dihydrofolate reductase (DFR) as a selectable marker conferringresistance to methotrexate. PCR is used to amplify the 35S promoter(−800 bp), intron 6 from the maize Adh1 gene (−550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250-bp fragment encodingthe E. coli dihydrofolate reductase type II gene is also amplified byPCR and these two PCR fragments are assembled with a SacI-PstI fragmentfrom pB1221 (Clontech) which comprises the pUC19 vector backbone and thenopaline synthase terminator. Assembly of these fragments generatespSOG19 which contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR gene and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus (MCMV) generates the vectorpSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistanceand have HindIII, SphI, PstI and EcoRI sites available for the cloningof foreign substances.

Vector Suitable for Chloroplast Transformation

For expression of a nucleotide sequence of the present invention inplant plastids, plastid transformation vector pPH143 (WO 97/32011,example 36, published Sep. 4, 1997; Novartis) is used. The nucleotidesequence is inserted into pPH143 thereby replacing the PROTOX codingsequence. This vector is then used for plastid transformation andselection of transformants for spectinomycin resistance. Alternatively,the nucleotide sequence is inserted in pPH143 so that it replaces theaadH gene. In this case, transformants are selected for resistance toPROTOX inhibitors.

III. Transformation Methods

Plants transformed in accordance with the present invention may bemonocots or dicots and include, but are not limited to, maize, wheat,barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear,quince, melon, canola, plum, cherry, peach, nectarine, apricot,strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya,mango, banana, soybean, tomato, sorghum, sugarcane, sugarbeet,sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice,potato, eggplant, cucumber, Arabidopsis thaliana, and woody plants suchas coniferous and deciduous trees, especially maize, wheat, orsugarbeet.

Once a desired DNA sequence has been transformed into a particular plantspecies, it may be propagated in that species or moved into othervarieties of the same species, particularly including commercialvarieties, using traditional breeding techniques.

Below are descriptions of representative techniques for transformingboth dicotyledonous and monocotyledonous plants, as well as arepresentative plastid transformation technique.

Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterium-based techniques and techniques that do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described by Paszkowski et al., EMBO J 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend of thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 forpCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

Another approach to transforming a plant cell with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792 all to Sanford et al (issued Jul. 31, 1990,Jul. 30, 1991, Mar. 31, 1992, respectively). Generally, this procedureinvolves propelling inert or biologically active particles at the cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the desired gene.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

Transformation of Monocotyledons

Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG (polyethylene glycol) or electroporationtechniques, and particle bombardment into callus tissue. Transformationscan be undertaken with a single DNA species or multiple DNA species(i.e. co-transformation) and both these techniques are suitable for usewith this invention. Co-transformation may have the advantage ofavoiding complete vector construction and of generating transgenicplants with unlinked loci for the gene of interest and the selectablemarker, enabling the removal of the selectable marker in subsequentgenerations, should this be regarded desirable. However, a disadvantageof the use of co-transformation is the less than 100% frequency withwhich separate DNA species are integrated into the genome (Schocher etal. Biotechnology 4: 1093-1096 (1986)).

Patent Applications EP 0 292 435 (issued Jul. 26, 1995 to Ciba Geigy),EP 0 392 225 (published Sep. 25, 1991; Ciba Geigy), and WO 93/07278(published Apr. 15, 1993; Ciba Geigy) describe techniques for thepreparation of callus and protoplasts from an elite inbred line ofmaize, transformation of protoplasts using PEG or electroporation, andthe regeneration of maize plants from transformed protoplasts.Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al.(Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, WO 93/07278 (published Apr. 15, 1993; Ciba Geigy) andKoziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques forthe transformation of elite inbred lines of maize by particlebombardment. This technique utilizes immature maize embryos of 1.5-2.5mm length excised from a maize ear 14-15 days after pollination and aPDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).Furthermore, WO 93/21335 (published Nov. 28, 1993; Plant GeneticSystems) describes techniques for the transformation of rice viaelectroporation. Patent Application EP 0 332 581 (issued Dec. 11, 1996to Ciba Geigy) describes techniques for the generation, transformationand regeneration of Pooideae protoplasts. These techniques allow thetransformation of Dactylis and wheat. Furthermore, wheat transformationhas been described by Vasil et al. (Biotechnology 10: 667-674 (1992))using particle bombardment into cells of type C long-term regenerablecallus, and also by Vasil et al. (Biotechnology 11: 1553-1558 (1993))and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particlebombardment of immature embryos and immature embryo-derived callus. Apreferred technique for wheat transformation, however, involves thetransformation of wheat by particle bombardment of immature embryos andincludes either a high sucrose or a high maltose step prior to genedelivery. Prior to bombardment, any number of embryos (0.75-1 mm inlength) are plated onto MS medium with 3% sucrose (Murashiga & Skoog,Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for inductionof somatic embryos, which is allowed to proceed in the dark. On thechosen day of bombardment, embryos are removed from the induction mediumand placed onto the osmoticum (i.e. induction medium with sucrose ormaltose added at the desired concentration, typically 15%). The embryosare allowed to plasmolyze for 2-3 h and are then bombarded. Twentyembryos per target plate is typical, although not critical. Anappropriate gene-carrying plasmid (such as pCIB3064 or pSG35) isprecipitated onto micrometer size gold particles using standardprocedures. Each plate of embryos is shot with the DuPont Biolistics®helium device using a burst pressure of ˜1000 psi using a standard 80mesh screen. After bombardment, the embryos are placed back into thedark to recover for about 24 h (still on osmoticum). After 24 hrs, theembryos are removed from the osmoticum and placed back onto inductionmedium where they stay for about a month before regeneration.Approximately one month later the embryo explants with developingembryogenic callus are transferred to regeneration medium (MS+1 mg/literNAA, 5 mg/liter GA), further containing the appropriate selection agent(10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in thecase of pSOG35). After approximately one month, developed shoots aretransferred to larger sterile containers known as “GA7s” which containhalf-strength MS, 2% sucrose, and the same concentration of selectionagent.

Transformation of monocotyledons using Agrobacterium has also beendescribed. See, WO 94/00977 (published Jan. 20, 1994; Japan Tobacco) andU.S. Pat. No. 5,591,616, (issued Jan. 7, 1997 to Japan Tobacco) both ofwhich are incorporated herein by reference.

Transformation of Plastids

Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ are germinated seven perplate in a 1″ circular array on T agar medium and bombarded 12-14 daysafter sowing with 1 μm tungsten particles (M10, Biorad, Hercules,Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially asdescribed (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). Bombardedseedlings are incubated on T medium for two days after which leaves areexcised and placed abaxial side up in bright light (350-500 μmolphotons/m²/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. andMaliga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/ml spectinomycindihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearingunderneath the bleached leaves three to eight weeks after bombardmentare subcloned onto the same selective medium, allowed to form callus,and secondary shoots isolated and subcloned. Complete segregation oftransformed plastid genome copies (homoplasmicity) in independentsubclones is assessed by standard techniques of Southern blotting(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digestedtotal cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5,346-349) is separated on 1% Tris-borate (TBE) agarose gels, transferredto nylon membranes (Amersham) and probed with ³²P-labeled random primedDNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment frompC8 containing a portion of the rps7/12 plastid targeting sequence.Homoplasmic shoots are rooted aseptically on spectinomycin-containingMS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) andtransferred to the greenhouse.

The invention will be further described by the following examples, whichare not intended to limit the scope of the invention in any manner.

EXAMPLE 1

The deduced amino acid sequence of Nov9x (SEQ ID NO: 1) was convertedinto a maize optimized nucleic acid sequence using the Wisconsin GCGanalysis program Backtranslate and the codon table for highly expressedmaize genes (see, e.g., U.S. Pat. No. 5,625,136). The synthetic gene wasprepared by Integrated DNA technologies, Inc. (Coralville, Iowa).

Nov9X phytase amino acid sequence (the 8 mutations are bolded andunderlined) (SEQ ID NO:1)MAQSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWPTWPVKLG E LT PRGGELIAYLGHY WRQRLVADGLL P K C GCPQSGQVAIIADVDERTRKTGE AFAAGLAPDCAITVHTQADTSSPDPLFNPLKTGVCQLDNANVTDAIL E RAG GS IADFTGH YQTAFRELERVLNFPQSNLCLKREKQDESCSLTQALPSELKVS AD CVSLTGAVSLASMLTEIFLLQQAQGMPEPGWGRITDSHQWNTLLSLHNAQ F DLLQRTPEVARSRATPLLDLIKTALTPHPPQKQAYGVTLPTSVLFIAGHD TNLANLGGALELNWTLPGQPDNTPPGGELVFERWRRLSDNSQWIQVSLVFQTL QQMRDKTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFTQIVNEARIPAC SL

Nov9X phytase maize-optimized nucleic acid sequence (SEQ ID NO:2) BamHIand SacI cloning sites (underlined) were included at the 5′ and 3′ ends,respectively. The start and stop codons are shown in bold.GGATCCACCATGGCGCAGTCCGAGCCGGAGCTGAAGCTGGAGTCCGTGGTGATCGTGTCCCGCCACGGCGTGCGCGCCCCGACCAAGGCCACCCAGCTCATGCAGGACGTGACCCCGGACGCCTGGCCGACCTGGCCGGTGAAGCTCGGCGAGCTGACCCCGCGCGGCGGCGAGCTGATCGCCTACCTCGGCCACTACTGGCGCCAGCGCCTCGTGGCCGACGGCCTCCTCCCGAAGTGCGGCTGCCCGCAGTCCGGCCAGGTGGCCATCATCGCCGACGTGGACGAGCGCACCCGCAAGACCGGCGAGGCCTTCGCCGCCGGCCTCGCCCCGGACTGCGCCATCACCGTGCACACCCAGGCCGACACCTCCTCCCCGGACCCGCTCTTCAACCCGCTCAAGACCGGCGTGTGCCAGCTCGACAACGCCAACGTGACCGACGCCATCCTGGAGCGCGCCGGCGGCTCCATCGGCGACTTCACCGGCCACTACCAGACCGCCTTCCGCGAGCTGGAGCGCGTGCTCAACTTCCCGCAGTCCAACCTCTGCCTCAAGCGCGAGAAGCAGGACGAGTCCTGCTCCCTCACCCAGGCCCTCCCGTCCGAGCTGAAGGTGTCCGCCGACTGCGTGTCCCTCACCGGCGCCGTGTCCCTCGCCTCCATGCTCACCGAAATCTTCCTCCTCCAGCAGGCCCAGGGCATGCCGGAGCCGGGCTGGGGCCGCATCACCGACTCCCACCAGTGGAACACCCTCCTCTCCCTCCACAACGCCCAGTTCGACCTCCTCCAGCGCACCCCGGAGGTGGCCCGCTCCCGCGCCACCCCGCTCCTCGACCTCATCAAGACCGCCCTCACCCCGCACCCGCCGCAGAAGCAGGCCTACGGCGTGACCCTCCCGACCTCCGTGCTCTTCATCGCCGGCCACGACACCAACCTCGCCAACCTCGGCGGCGCCCTGGAGCTGAACTGGACCCTCCCGGGCCAGCCGGACAACACCCCGCCGGGCGGCGAGCTGGTGTTCGAGCGCTGGCGCCGCCTCTCCGACAACTCCCAGTGGATTCAGGTGTCCCTCGTGTTCCAGACCCTCCAGCAGATGCGCGACAAGACCCCGCTCTCCCTCAACACCCCGCCGGGCGAGGTGAAGCTCACCCTCGCCGGCTGCGAGGAGCGCAACGCCCAGGGCATGTGCTCCCTCGCCGGCTTCACCCAGATCGTGAACGAGGCCCGCATCCCGGCCTGCTCCCTCTAATAGAGCTC

A. Preparation of Expression Cassettes Having a Maize-Optimized PhytaseGene

The following Nov9x cassettes were constructed to express the Nov9Xphytase in maize seed with various targeting signals. The Nov9x codingsequence has a BamHI cloning site at the 5′ end and a SacI cloning siteat the 3′ end.

pNOV4054 comprises the γ-zein N-terminal signal sequence(MRVLLVALALLALAASATS) (SEQ ID NO:3) fused to the synthetic Nov9X phytasefor targeting to the endoplasmic reticulum and secretion into theapoplast (Torrent et al., 1997). The first residue after the signalsequence is Ala. This replaces Met1 in Nov9x.

pNOV4054 phytase fusion amino acid sequence (SEQ ID NO:4) (γ-zeinsequence is in bold) MRVLLVALALLALAASATSAAQSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWPTWPVKLGELTPRGGELIAYLGHYWRQRLVADGLLPKCGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDPLFNPLK TGVCQLDNANVTDAILERAGGSIADFTGHYQTAFRELERVLNFPQSNLCLKR EKQDESCSLTQALPSELKVSADCVSLTGAVSLASMLTEIFLLQQAQGMPEPG WGRITDSHQWNTLLSLHNAQFDLLQRTPEVARSRATPLLDLIKTALTPHPP QKQAYGVTLPTSVLFIAGHDTNLANLGGALELNWTLPGQPDNTPPGGELVFE RWRRLSDNSQWIQVSLVFQTLQQMRDKTPLSLNTPPGEVKLTLAGCEERNA QGMCSLAGFTQIVNEARIPACSL

pNOV4054 phytase fusion nucleotide sequence (SEQ ID NO: 5) showingflanking 5′ BamHI and 3′ SacI cloning sites (underlined). Start and stopcodons are indicated in bold type.GGATCCACCATGAGGGTGTTGCTCGTTGCCCTCGCTCTCCTGGCTCTCGCTGCGAGCGCCACCAGCGCTGCGCAGTCCGAGCCGGAGCTGAAGCTGGAGTCCGTGGTGATCGTGTCCCGCCACGGCGTGCGCGCCCCGACCAAGGCCACCCAGCTCATGCAGGACGTGACCCCGGACGCCTGGCCGACCTGGCCGGTGAAGCTCGGCGAGCTGACCCCGCGCGGCGGCGAGCTGATCGCCTACCTCGGCCACTACTGGCGCCAGCGCCTCGTGGCCGACGGCCTCCTCCCGAAGTGCGGCTGCCCGCAGTCCGGCCAGGTGGCCATCATCGCCGACGTGGACGAGCGCACCCGCAAGACCGGCGAGGCCTTCGCCGCCGGCCTCGCCCCGGACTGCGCCATCACCGTGCACACCCAGGCCGACACCTCCTCCCCGGACCCGCTCTTCAACCCGCTCAAGACCGGCGTGTGCCAGCTCGACAACGCCAACGTGACCGACGCCATCCTGGAGCGCGCCGGCGGCTCCATCGCCGACTTCACCGGCCACTACCAGACCGCCTTCCGCGAGCTGGAGCGCGTGCTCAACTTCCCGCAGTCCAACCTCTGCCTCAAGCGCGAGAAGCAGGACGAGTCCTGCTCCCTCACCCAGGCCCTCCCGTCCGAGCTGAAGGTGTCCGCCGACTGCGTGTCCCTCACCGGCGCCGTGTCCCTCGCCTCCATGCTCACCGAAATCTTCCTCCTCCAGCAGGCCCAGGGCATGCCGGAGCCGGGCTGGGGCCGCATCACCGACTCCCACCAGTGGAACACCCTCCTCTCCCTCCACAACGCCCAGTTCGACCTCCTCCAGCGCACCCCGGAGGTGGCCCGCTCCCGCGCCACCCCGCTCCTCGACCTCATCAAGACCGCCCTCACCCCGCACCCGCCGCAGAAGCAGGCCTACGGCGTGACCCTCCCGACCTCCGTGCTCTTCATCGCCGGCCACGACACCAACCTCGCCAACCTCGGCGGCGCCCTGGAGCTGAACTGGACCCTCCCGGGCCAGCCGGACAACACCCCGCCGGGCGGCGAGCTGGTGTTCGAGCGCTGGCGCCGCCTCTCCGACAACTCCCAGTGGATTCAGGTGTCCCTCGTGTTCCAGACCCTCCAGCAGATGCGCGACAAGACCCCGCTCTCCCTCAACACCCCGCCGGGCGAGGTGAAGCTCACCCTCGCCGGCTGCGAGGAGCGCAACGCCCAGGGCATGTGCTCCCTCGCCGGCTTCACCCAGATCGTGAACGAGGCCCGCATCCCGGCCTGCTCCCTCTAATAGAGCTC

pNOV4058 comprises the γ-zein N-terminal signal sequence fused to thesynthetic Nov9X phytase with a C-terminal addition of the sequenceSEKDEL for targeting to and retention in the endoplasmic reticulum(Munro and Pelham, 1987). The first residue after the signal sequence isAla. This replaces Met1 in Nov9x.

pNOV4058 phytase fusion amino acid sequence (SEQ ID NO:6) (γ-zein atN-terminus and SEKDEL sequence at C-terminus are shown in bold):MRVLLVALALLALAASATSAAQSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWPTWPVKLGELTPRGGELIAYLGHYWRQRLVADGLLPKCGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDPLFNPLK TGVCQLDNANVTDAILERAGGSIADFTGHYQTAFRELERVLNFPQSNLCLKR EKQDESCSLTQALPSELKVSADCVSLTGAVSLASMLTEIFLLQQAQGMPEPG WGRITDSHQWNTLLSLHNAQFDLLQRTPEVARSRATPLLDLIKTALTPHPP QKQAYGVTLPTSVLFIAGHDTNLANLGGALELNWTLPGQPDNTPPGGELVFE RWRRLSDNSQWIQVSLVFQTLQQMRDKTPLSLNTPPGEVKLTLAGCEERNA QGMCSLAGFTQIVNEARIPACSLSEKDEL

pNOV4058 phytase fusion nucleotide sequence (SEQ ID NO:7) showingflanking 5′ BamHI and 3′ SacI cloning sites (underlined). Sequencesencoding the gamma-zein signal sequence and SEKDEL signal are shown inbold type. GGATCCACCATGAGGGTGTTGCTCGTTGCCCTCGCTCTCCTGGCTCTCGCTGCGAGCGCCACCAGCGCTGCGCAGTCCGAGCCGGAGCTGAAGCTGGAGTCCGTGGTGATCGTGTCCCGCCACGGCGTGCGCGCCCCGACCAAGGCCACCCAGCTCATGCAGGACGTGACCCCGGACGCCTGGCCGACCTGGCCGGTGAAGCTCGGCGAGCTGACCCCGCGCGGCGGCGAGCTGATCGCCTACCTCGGCCACTACTGGCGCCAGCGCCTCGTGGCCGACGGCCTCCTCCCGAAGTGCGGCTGCCCGCAGTCCGGCCAGGTGGCCATCATCGCCGACGTGGACGAGCGCACCCGCAAGACCGGCGAGGCCTTCGCCGCCGGCCTCGCCCCGGACTGCGCCATCACCGTGCACACCCAGGCCGACACCTCCTCCCCGGACCCGCTCTTCAACCCGCTCAAGACCGGCGTGTGCCAGCTCGACAACGCCAACGTGACCGACGCCATCCTGGAGCGCGCCGGCGGCTCCATCGCCGACTTCACCGGCCACTACCAGACCGCCTTCCGCGAGCTGGAGCGCGTGCTCAACTTCCCGCAGTCCAACCTCTGCCTCAAGCGCGAGAAGCAGGACGAGTCCTGCTCCCTCACCCAGGCCCTCCCGTCCGAGCTGAAGGTGTCCGCCGACTGCGTGTCCCTCACCGGCGCCGTGTCCCTCGCCTCCATGCTCACCGAAATCTTCCTCCTCCAGCAGGCCCAGGGCATGCCGGAGCCGGGCTGGGGCCGCATCACCGACTCCCACCAGTGGAACACCCTCCTCTCCCTCCACAACGCCCAGTTCGACCTCCTCCAGCGCACCCCGGAGGTGGCCCGCTCCCGCGCCACCCCGCTCCTCGACCTCATCAAGACCGCCCTCACCCCGCACCCGCCGCAGAAGCAGGCCTACGGCGTGACCCTCCCGACCTCCGTGCTCTTCATCGCCGGCCACGACACCAACCTCGCCAACCTCGGCGGCGCCCTGGAGCTGAACTGGACCCTCCCGGGCCAGCCGGACAACACCCCGCCGGGCGGCGAGCTGGTGTTCGAGCGCTGGCGCCGCCTCTCCGACAACTCCCAGTGGATTCAGGTGTCCCTCGTGTTCCAGACCCTCCAGCAGATGGGCGACAAGACCCCGCTCTCCCTCAACACCCCGCCGGGCGAGGTGAAGCTCACCCTCGCCGGCTGCGAGGAGCGCAACGCCCAGGGCATGTGCTCCCTCGCCGGCTTCACCCAGATCGTGAACGAGGCCCGCATCCCGGCCTGCTCCCTCTCCGAGAAGGACGAGCTGTAATA GAGCTCB. Isolation of Promoters for Endosperm-Specific Expression in Maize

The promoter from the Zea mays γ-zein gene (obtained from Dr. BrianLarkins) is amplified as a 673 bp fragment from plasmid pGZ27.3. Theγ-zein promoter has been shown to be endosperm specific (Torrent et al.,1997). HindIII and BamHI cloning sites were introduced at the 5′ and 3′ends, respectively. These sites are underlined.

Zea mays γ-zein promoter nucleic acid sequence (SEQ ID NO:8)AAGCTTCGATCATCCAGGTGCAACCGTATAAGTCCTAAAGTGGTGAGGAACACGAAACAACCATGCATTGGCATGTAAAGCTCCAAGAATTTGTTGTATCCTTAACAACTCACAGAACATCAACCAAAATTGCACGTCAAGGGTATTGGGTAAGAAACAATCAAACAAATCCTCTCTGTGTGCAAAGAAACACGGTGAGTCATGCCGAGATCATACTCATCTGATATACATGCTTACAGCTCACAAGACATTACAAACAACTCATATTGCATTACAAAGATCGTTTCATGAAAAATAAAATAGGCCGGACAGGACAAAAATCCTTGACGTGTAAAGTAAATTTACAACAAAAAAAAAGCCATATGTCAAGCTAAATCTAATTCGTTTTACGTAGATCAACAACCTGTAGAAGGCAACAAAACTGAGCCACGCAGAAGTACAGAATGATTCCAGATGAACCATCGACGTGCTACGTAAAGAGAGTGACGAGTCATATACATTTGGCAAGAAACCATGAAGCTGCCTACAGCCGTCTCGGTGGCATAAGAACACAAGAAATTGTGTTAATTAATCAAAGCTATAAATAACGCTCGCATGCCTGTGCACTTCTCCATCACCACCACTGGGTCTTCAGACCATTAGCTTTATCTACTCCAGAGCGCAGAAGAACCCGATCGACAGGATCCC. Isolation of Promoters for Embryo-Specific Expression in Maize

The promoter and 5′ noncoding region of the major maize embryo globulin,glob1, was amplified as a 1427 base pair fragment from maize genomic DNAusing primers designed from Genbank accession L22344. The globulinpromoter has been shown to be primarily embryo-specific (Belanger andKriz, 1989). HindIII and BamHI cloning sites were introduced at the 5′and 3′ ends, respectively. These sites are underlined.

Zea mays globulin1 promoter nucleic acid sequence (SEQ ID NO:9)AAGCTTAGTGCCATCCTTGGACACTCGATAAAGTATATTTTATTTTTTTT ATTTTGCCAACCAAACTTTTTGTGGTATGTTCCTACACTATGTAGATCTAC ATGTACCATTTTGGCACAATTACATATTTACAAAAATGTTTTCTATAAATATTAGATTTAGTTCGTTTATTTGAATTTCTTCGGAAAATTCACATTTAAAC TGCAAGTCACTCGAAACATGGAAAACCGTGCATGCAAAATAAATGATATGCATGTTATCTAGCACAAGTTACGACCGATTTCAGAAGCAGACCAGAATCTTCAAGCACCATGCTCACTAAACATGACCGTGAACTTGTTATCTAGTTGTTTAAAAATTGTATAAAACACAAATAAAGTCAGAAATTAATGAAACTTGTCCACATGTCATGATATCATATATAGAGGTTGTGATAAAAATTTGATAATGTTTCGGTAAAGTTGTGACGTACTATGTGTAGAAACCTAAGTGACCTACACATAAAATCATAGAGTTTCAATGTAGTTCACTCGACAAAGACTTTGTCAAGTGTCCGATAAAAAGTACTCGACAAAGAAGCCGTTGTCGATGTACTGTTCGTCGAGATCTCTTTGTCGAGTGTCACACTAGGCAAAGTCTTTACGGAGTGTTTTTCAGGCTTTGACACTCGGCAAAGCGCTCGATTCCAGTAGTGACAGTAATTTGCATCAAAAATAGCTGAGAGATTTAGGCCCCGTTTCAATCTCACGGGATAAAGTTTAGCTTCCTGCTAAACTTTAGCTATATGAATTGAAGTGCTAAAGTTTAGTTTCAATTACCACCATTAGCTCTCCTGTTTAGATTACAAATGGCTAAAAGTAGCTAAAAAATAGCTGCTAAAGTTTATCTCGCGAGATTGAAACAGGGCCTTAAAATGAGTCAACTAATAGACCAACTAATTATTAGCTATTAGTCGTTAGCTTCTTTAATCTAAGCTAAAACCAACTAATAGCTTATTTGTTGAATTACAATTAGCTCAACGGAATTCTCTGTTTTTTCTATAAAAAAAGGGAAACTGCCCCTCATTTACAGCAAATTGTCCGCTGCCTGTCGTCCAGATACAATGAACGTACCTAGTAGGAACTCTTTTACACGCTCGGTCGCTCGCCGCGGATCGGAGTCCCAGGAACACGACACCACTGTGTAACACGACAAAGTCTGCTCAGAGGCGGCCACACCCTGGCGTGCACCGAGCCGGAGCCCGGATAAGCACGGTAAGGAGAGTACGGCGGGACGTGGCGACCCGTGTGTCTGCTGCCACGCAGCCTTCCTCCACGTAGCCGCGCGGCCGCGCCACGTACCAGGGCCCGGCGCTGGTATAAATGCGCGCTACCTCCGCTTTAGTTCTGCATACAGTCAACCTAACACACCCGAGCATATCACAGTGGGATCCD. Construction of Plant Transformation Vectors for the Maize-OptimizedPhytase Gene

Binary vectors for maize transformation were constructed in two steps.In the first step, three fragments were fused to generate an expressioncassette. The expression cassette consisted of a HindIII-BamHI promotercassette (sections B & C above) fused to a BamHI-SacI Nov9x cassette(section A above) fused to a SacI-KpnI terminator cassette. Theterminator cassette included an inverted PEPC intron. The expressioncassette was then transferred as a HindIII-KpnI fragment into the binaryvector pNOV2117, which contains the phosphomannose isomerase (PMI) geneallowing for selection of transgenic cells with mannose.

A summary of the binary vectors prepared for plant transformation isshown in Table 1. The six vectors were introduced into maize.

The Nov9X binary vectors listed in Table 1 all contain the sameterminator cassette with a PEPC intron. Vectors pNOV4051, 4055, and 4059contain the globulin promoter cassette, and vectors pNOV4053, 4057, and4061 contain the gamma-zein promoter cassette. Vectors pNOV4051 and 4053contain the Nov9X sequence shown in SEQ ID NO:2 with cytoplasmictargeting. Vectors pNOV4055 and 4057 contain the Nov9X cassette frompNOV4054 with apoplast targeting. Vectors pNOV4059 and 4061 contain theNov9X cassette from pNOV4058 with ER retention. TABLE 1 promoter Vector(source) signal seq. gene Crop Predicted localization PNOV4051 globulinnone NOV9X maize embryo cytoplasm PNOV4053 gamma-zein none NOV9X maizeendosperm cytoplasm PNOV4055 globulin gamma-zein NOV9X maize embryoapoplast PNOV4057 gamma-zein gamma-zein NOV9X maize endosperm apoplastPNOV4059 globulin gamma-zein NOV9X-SEKDEL maize embryo ER PNOV4061gamma-zein gamma-zein NOV9X-SEKDEL maize endosperm ER

The vectors pNOV4057 and pNOV4059 have been deposited in theAgricultural Research Culture Collection (NRRL), 1815 N, UniversityStreet, Peoria, Ill. 61604, USA, as International Depositary Authorityas established under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure, Accession numbers NRRL B-30537, and NRL B-30538,respectively, on Dec. 28, 2001.

EXAMPLE 2

Genetic Modification of Maize and Wheat

The synthetic Nov9X gene inserted into an appropriate vector asdescribed above was introduced into maize by Agrobacterium-mediatedtransformation and into wheat by biolistic transformation. Stabletransformants were regenerated from tissue cultured on selective mediausing the Positech system in accordance with U.S. Pat. Nos. 5,767,378and 5,994,629.

Agrobacterium-Mediated Transformation of Maize

Transformation of immature maize embryos is performed essentially asdescribed in Negrotto et al., (2000) Plant Cell Reports 19: 798-803.Various media constituents described therein can be substituted.

Transformation Plasmids and Selectable Marker

The genes used for transformation are cloned into a vector suitable formaize transformation as described above. Vectors used contain thephosphomannose isomerase (PMI) gene (Negrotto et al. (2000) Plant CellReports 19: 798-803) as a selectable marker.

Preparation of Agrobacterium tumefaciens

Agrobacterium strain LBA4404 (pSB1) containing the plant transformationplasmid is grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl(5 g/L), 15 g/l agar, pH 6.8) solid medium for 2 to 4 days at 28° C.Approximately 0.8×10⁹ Agrobacteria are suspended in LS-inf mediasupplemented with 100 μM acetosyringone (As) (LSAs medium) (Negrotto etal., (2000) Plant Cell Rep 19: 798-803). Bacteria are pre-induced inthis medium for 30-60 minutes.

Inoculation

Immature embryos from A188 or other suitable maize genotypes are excisedfrom 8-12 day old ears into liquid LS-inf +100 μM As (LSAs). Embryos arevortexed for 5 seconds and rinsed once with fresh infection medium.Infection media is removed and Agrobacterium solution is then added andembryos are vortexed for 30 seconds and allowed to settle with thebacteria for 5 minutes. The embryos are then transferred scutellum sideup to LSAs medium and cultured in the dark for two to three days.Subsequently, between 20 and 25 embryos per petri plate are transferredto LSDc medium supplemented with cefotaxime (250 mg/l) and silvernitrate (1.6 mg/l) (Negrotto et al. 2000) and cultured in the dark for28° C. for 10 days.

Selection of Transformed Cells and Regeneration of Transformed Plants

Immature embryos producing embryogenic callus are transferred toLSD1M0.5S medium (LSDc with 0.5 mg/l 2,4-D instead of Dicamba, 10 g/lmannose, 5 g/l sucrose and no silver nitrate). The cultures are selectedon this medium for 6 weeks with a subculture step at 3 weeks. Survivingcalli are transferred either to LSD1M0.5S medium to be bulked-up or toReg1 medium (as described in Negrotto et al., 2000). Following culturingin the light (16 hour light/8 hour dark regiment), green tissues arethen transferred to Reg2 medium without growth regulators (as describedin Negrotto et al. 2000) and incubated for 1-2 weeks. Plantlets aretransferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.)containing Reg3 medium (as described in Negrotto et al. 2000) and grownin the light. Plants that are PCR positive for the Nov9X expressioncassette are transferred to soil and grown in the greenhouse.

DNA Analysis

The presence of the Nov9X gene was determined by +/−PCR assay or by aTaqman copy number assay. The presence of the PMI selective marker wasdetermined by a Taqman copy number assay. The presence of thespectinomycin resistance gene selective marker was determined by +/−PCRassay.

EXAMPLE 3

Protein Extraction from Corn Seeds

Ears from first and second generation transgenic maize plants wereharvested at 24-40 days or about 30 days after pollination (DAP). Freshkernels and isolated endosperm were pulverized in water at roomtemperature. Proteins were extracted in water using a mortar and pestle.

Alternatively, kernels were dried on the cob at 105° F. for 5 days.Dried kernels were pulverized to yield a flour using a Kleco tissuepulverizer at room temperature for 20-30 seconds. Proteins wereextracted from 100 mg flour by addition of 1 ml buffer and incubationfor 20 min at room temperature.

Insoluble material was removed by centrifugation for 10 min at 4° C.Extracts were kept on ice. Protein concentration was determined usingthe ADV01 protein assay reagent (Cytoskeleton, Denver, Colo.) with BSAas the standard. Protein assays were performed in 96-well plates and 310μl reactions. Absorbance was measured at 595 nm using a SpectraMaxPlusplate reader (Molecular Devices). Typically 10 μl of a 10-fold dilutionof the extract was diluted further with 300 μl of ADV01 reagent.

Enzyme Assay

Estimation of Phytase Activity

Determination of phytase activity, based upon the estimation ofinorganic phosphate released on hydrolysis of phytic acid, can beperformed at 37° C. following the method described by Engelen et al.(2001). One unit of enzyme activity is defined as the amount of enzymethat liberates 1 μmol of inorganic phosphate per minute under assayconditions. For example, phytase activity may be measured by incubating2.0 ml of the enzyme preparation with 4.0 ml of 9.1 mM sodium phytate in250 mM sodium acetate buffer pH 5.5, supplemented with 1 mM CaCl₂ for 60minutes at 37° C. After incubation, the reaction is stopped by adding4.0 ml of a color-stop reagent consisting of equal parts of a 10% (w/v)ammonium molybdate and a 0.235% (w/v) ammonium vanadate stock solution.Phosphate released is measured against a set of phosphate standardsspectrophotometrically at 415 nm. Phytase activity is calculated byinterpolating the A₄₁₅ nm absorbance values obtained for phytasecontaining samples using the generated phosphate standard curve.Alternatively, a phytase activity curve generated by using astandardized phytase reference whose activity is certified by themanufacturer may be used in place of a phosphate standard curve todetermine enzymatic activity. Specific activity can be expressed inunits of enzyme activity per mg of protein.

Alternatively, phytase activity may be measured according to theprocedure of Wyss et al., 1999 with modifications. Assays were performedin 1.5 ml tubes and 96-well microplates. The tube assays were started bymixing 5 μl 1 M sodium acetate (pH 4.5), 10 μl 10 mM phytic acid (SigmaCat. # P8810), 5 μl distilled water, and 5 μl diluted extract. Thereaction was incubated at 37° C. for 10 min, after which the tubes wereplaced on ice and the reaction quenched immediately by addition of 25 μl15% trichloroacetic acid (TCA). The quenched reaction was diluted with450 μl distilled water.

Colorimetric determination of phosphate concentration was initiated byaddition of 500 μl of the colorimetric reagent (0.6 M sulfuric acid, 2%ascorbate, 0.5% ammonium molybdate) and incubation at 50° C. for 15 min.Phosphate concentration was determined by measuring absorbance at 800 nmand comparison to a standard curve of potassium phosphate.

Assays in microplates (flat bottom, 300 μl well volume) were started bymixing 20 μl 1 M sodium acetate, 40 μl 10 mM phytic acid, and 40 μl ofdiluted extract. The plate was kept on ice during mixing. The plate wassealed with foil and placed on a plate heater (Boekel-Grant PH-100) at37° C. for 10 min. The plate was transferred to ice and the reactionswere quenched by addition of 100 μl 15% TCA. 15 μl of the quenchedreaction was diluted with 135 μl distilled water in a second plate, andthe calorimetric reaction was initiated by addition of 150 μl of thecolorimetric reagent. The second plate included phosphate standards. Theplate was sealed with foil and incubated on the plate heater at 50° C.for 15 min. Absorbance at 820 nm was measured for duplicate standardsand samples.

SDS-PAGE and Western Blot Analyses

Electrophoresis sample buffer and running buffer were according toLaemmli, 1970. Samples were boiled for 2 minutes prior toelectrophoresis using precast NOVEX Tris-Glycine gels (Invitrogen).Electrophoretic transfers were performed using the NOVEX Mini-Cellsystem (Invitrogen) using the manufacturer's protocol and bufferrecipes. The transfer buffer contained 12 mM Tris, 96 mM glycine, and20% methanol. Polypeptides were transferred to nitrocellulose (NOVEXLC2000) for 1-2 hours at 25 V and room temperature. Membranes wereblocked for 15 min at room temperature by incubation in 30 mM Tris-HCl(pH 10.2), 150 mM NaCl, and 0.05% Tween-20 (TBST) supplemented with 3%BSA.

The primary immune serum from goat was obtained from Duncroft, Inc.(Lovettsville, Va.). The goat was inoculated with recombinant Nov9Xphytase extracted from E. coli (obtained from Diversa). Blots wereincubated with primary antibody (1:2000 dilution in TBST) for 1 hour atroom temperature followed by 3 5-minute washes with TBST. The procedurewas repeated using secondary antibody (mouse anti-goat IgG) diluted1:50,000 in TBST. Blots were developed using enhanced chemiluminescence(Pierce SuperSignal Ultra).

EXAMPLE 3

Accumulation of Nov9X Phytase in Maize Seed from First GenerationTransgenic Plants Transformed with Vectors pNOV4057 and pNOV4061.

An exemplary formulation of phytase enzyme produced in corn seed is inthe form of a liquid extract. Here, the enrichment of Nov9X phytase bydegermination, water extraction, heating, and centrifugation isdescribed.

Fresh kernels from two of the first generation transgenic plants toproduce mature ears were analyzed for phytase activity. The plants werederived from embryos that had been transformed with plasmids pNOV4057(event 305A13) and pNOV4061 (event 305B11). Fresh kernels of the sameage were obtained from a third ear that did not contain the Nov9Xphytase gene. This control plant (event 264A8C#14) contained the vectorpNOV4314, which encodes a heterologous enzyme. Six kernels and sixendosperm from each of the three ears were pooled and crushed using amortar and pestle. A total of 10 ml ddH₂0 was added to the kernels. Thesoluble fractions of the suspensions were analyzed for total protein andphytase activity.

Results of phytase assays of kernel and endosperm extracts are shown inFIG. 1. The assay measures phosphate concentration in the reaction aftera 10 minute incubation at 37° C. Extracts from the negative controltissue are included in order to determine the level of endogenousphosphate (FIG. 1, top). This level of background phosphate isreproducible based on analysis of several extractions of flour fromcontrol samples (data not shown). After correcting for backgroundphosphate, phytase activity was calculated for the two plants containingthe Nov9X phytase gene. The phytase activity is reported as total unitsextracted from 6 kernels and is shown in FIG. 1, bottom. The differencesin total phytase activity between kernels and endosperm from the sameear is due to segregation of the transgene(s).

A portion of each of the endosperm extracts analyzed in FIG. 1 washeated at 60° C. for 30 min. As shown in FIG. 2A, the phytase activityin these extracts was unaffected by heating. Samples of unheated andheated endosperm extracts were analyzed by SDS-PAGE (FIG. 2B). Thecalculated size of the Nov9X gene product is 43 kDa. A protein in therange of 40-50 kD was abundant in the extract of 305B11. This proteinremained soluble during heating and was the most abundant protein in thesoluble fraction of the heated endosperm extract. A band in this regionof the gel was identified by Western blot analysis using antiserum froma goat inoculated with Nov9X produced in E. coli. This band is a goodcandidate for the recombinant phytase enzyme.

An additional source of enzyme for a liquid formulation is to use driedseed or flour obtained from dried seed. The seed may be milled toproduce flour. Seed harvested 30 DAP (days after pollination) fromseveral plants were dried for five days at 105° F. Dried seed werepulverized and proteins were extracted in buffer as described above. Acomparison of phytase activity in kernel extracts of 22 plants is shownin FIG. 3. Four plants with the highest levels of phytase activity froman initial screening of 24 plants were included in the analysis.

Another exemplary formulation of phytase enzyme produced in corn seed isas cracked or milled grain, or flour. Phytase-containing flour can beadded directly to animal feed as a supplement, or it can be processedfurther, for example to a pelleted form. The following experimentcompares the phytase activity of a liquid extract with flour.

Twenty milligrams (mg) flour was incubated in 200 μl buffer for 20minutes at room temperature. One set of tubes was then chilled on icewhile the insoluble material in the second set was removed bycentrifugation at 4° C. The total suspension from the first set and thetotal supernatant fraction from the second set of tubes were thendiluted to a final volume of 5 ml with extraction buffer. Phytic acid(10 ml 0.1 M) and sodium acetate (10 ml 0.5 M, pH 5) were added and thetubes were incubated for 10 min at 37° C. The final concentration of 40mM phytic acid represents a 10-fold increase over that used in thestandard assay described above. The reactions were chilled on ice andimmediately quenched by addition of an equal volume (25 ml) of 15% TCA.Insoluble material was removed by centrifugation (15 min, 4° C., 3,700rpm) and a portion of the supernatant fraction was used to measurephosphate concentration as described above.

The phytase activity in flour extracts and flour suspensions wascompared in order to determine the efficiency of the buffer extractionprocedure. Whole kernel flour (20 mg) was incubated with 200 μl ofextraction buffer and the total phytase activity of the supernatantfraction and flour suspension was determined. The results are shown inFIG. 4. Total phosphate concentrations in equivalent portions of thereactions are shown in the top graph. Event 266B-2E is a negativecontrol that does not contain recombinant phytase. These samples wereassayed to determine levels of endogenous phosphate. Events 305B-20A and305A-24A are transgenic phytase events transformed with vectors pNOV4061and pNOV4057, respectively. The yellow bars indicate phosphateconcentrations in reactions containing the soluble extract and the bluebars indicate phosphate levels in reactions containing the floursuspension. For both transgenic events additional phosphate wasliberated in the presence of transgenic flour.

The graphs in the bottom of FIG. 4 compare phytase activity in thesoluble extracts (yellow) and flour suspensions (blue) of the twoevents. For events 305B-20A (pNOV4061) and 305A-24A (pNOV4057)additional activity of 50% and 80%, respectively, remained associatedwith the flour.

EXAMPLE 4

Endosperm-Specific Expression of Nov9x Phytase in T2 Seed

Ears from two T2 plants containing construct pNOV4057 were harvested at24 and 26 days after pollination. Ten kernels from each plant weredissected into embryos, endosperm, and hulls. Ten endosperm and tenembryos were combined and pulverized with a mortar and pestle in 8 mldistilled water at room temperature. Insoluble material was removed bycentrifugation. The specific activity of supernatant fractions ofendosperm and embryo samples were determined. As shown in FIG. 5, thespecific activity of phytase in extracts of transgenic endospermexceeded 300. By contrast the specific activity of extracts oftransgenic embryos was less than 10. This barely detectable activity wasprobably due to contamination of embryos with small pieces of endosperm.

Nov9X Phytase is Highly Enriched by Heating Water Extracts of Fresh T2Endosperm

As disclosed above, an exemplary formulation of phytase enzyme producedin corn seed is in the form of a liquid extract. Here, the enrichment ofNov9X phytase by degermination, water extraction, heating, andcentrifugation is described.

As described above, kernels were degerminated manually and endospermwere pulverized in water using a mortar and pestle. The water extractsof fresh endosperm were heated at 60° C. for 30 min, and insolublematerial was removed by centrifugation. Heated and unheated samples wereanalyzed by SDS-PAGE (gel not shown). A protein of approximately 45 kD(arrow), the predicted size of Nov9X phytase, was enriched in the heatedsamples. This protein was not present in the control extracts and wasthe most abundant protein in the transgenic extracts. We conclude thatthis protein is Nov9X phytase. The total phytase activity was the samein unheated and heated samples (FIG. 6A), demonstrating that Nov9Xphytase produced in corn is thermostable. The specific activity of theheated samples increased up to two-fold (FIG. 6B). These resultsdemonstrate a possible strategy for preparing a liquid formulation ofNov9X phytase from corn seed. The same approach using dried endosperm orwhole kernel flour should also produce an extract highly enriched forthe recombinant enzyme.

Production and Processing of T2 Greenhouse Seed for Poultry FeedingTrials

As disclosed above, an exemplary formulation of phytase enzyme producedin corn seed is as cracked or milled grain, or flour. Phytase-containingflour can be added directly to animal feed as a supplement, or it can beprocessed further, for example to a pelleted form.

Corn plants were grown in the greenhouse from T1 seed from sevendifferent T0 transgenic events. All plants contained one or more copiesof the Nov9X gene as determined by Taqman analysis. These T1 plants wereeither selfed or fertilized with pollen from a sib or from JHAF031. Earswere harvested at about 30 days after pollination and dried for 5 daysat 105° F. The T2 seed from different ears were pooled for each of thesix transgenic events. Seeds were milled at room temperature in a fumehood using The Kitchen Mill (K-TEC) set to the finest grind. Batches ofseed ranged from 66 g to 398 g. Flour was transferred immediately fromthe mill chamber to sealed plastic bags in a fume hood. The temperatureof the flour was measured immediately after transfer to the plastic bagsand did not exceed 41° C. Flour was stored at 4° C.

T2 maize seed derived from six different T0 transgenic events werepooled and milled as described above. A sample of seed from maize inbredA188 was also processed for use as a negative control. The level ofphytase activity in maize flour was determined using the proceduredescribed above for protein extraction from dried seed and phytaseassays using a microplate format. Protein extractions were performedusing duplicate flour samples of about 100 mg.

The six transgenic events selected for seed increase are listed in Table2. They were derived from plasmids pNOV4057 and pNOV4061, which encodeapoplast-targeted and ER-retained forms of Nov9X phytase, respectively.The fraction of seed derived from selfed and crossed plants variedconsiderably between event pools. Also most T1 plants were nothomozygous for Nov9X. These factors make it impossible to correlateenzyme yields with gene copy number in the final T2 seed. The level ofextractable phytase activity measured in units per kg flour ranged from325,000 to 1,300,000 (Table 2).

Table 2 shows the yield of buffer-extractable phytase activity in flourfrom milled T2 greenhouse seed. A188 is a non-transgenic inbred. TABLE 2Binary Subcellular vector targeting Event Flour (g) Average StDevUnits/kg A188 control 368 0 0 PNOV4061 ER 305B-20A 398 425,666 63,2281,069,511 pNOV4061 ER 305B-11A 347 298,197 2,793 859,359 pNOV4061 ER305B-5A 204 141,200 15,966 692,157 pNOV4057 apoplast 305A-20A 66 21,4671,796 325,255 PNOV4057 apoplast 305A-11A 313 231,439 39,498 739,423PNOV4057 apoplast 305A-24A RT 200 256,782 24,510 1,283,910 pNOV4057apoplast 305A-24A cold 227 296,674 12,285 1,306,934

Milling corn at room temperature does not result in decreased phytaseactivity. The pooled seed derived from event 305A-24A was divided inhalf before milling. One batch was milled at room temperature. Thetemperature of the flour from this batch immediately after transfer was38° C. The other batch was chilled, along with the mill, in a 4° C. coldroom for 45 minutes. This batch was then milled in the cold room. Thesealed mill was then moved to the fume hood at ambient temperature andthe flour was transferred to a plastic bag. The temperature of thiscold-milled flour measured immediately after transfer was 24° C. Asshown in the bottom two rows of Table 2, milling chilled seeds in thecold did not improve yields of extractable phytase activity. The phytaseyield of seeds derived from event 305A-24A that were milled at roomtemperature (RT) was 1,283,910 units/kg compared to 1,306,934 units/kgfor chilled seeds from the same pool milled in the cold.

Table 3 shows the total flour and phytase yields when the separateevents are pooled by vector. These data are the sums of theevent-specific data in Table 2. The total yields of extractable phytaseactivity for both the apopast-targeted and ER-retained forms of theenzyme were greater than 800,000 total units from less than 1 kg flour.The flour described here is an example of a possible formulation ofNov9X phytase for use as a feed additive. Table 3 provides the phytaseyields from milled T2 greenhouse seed pooled for apoplast-targeted andER-retained forms. TABLE 3 Binary Subcellular vector targeting UnitsFlour (g) Units/kg pNOV4061 ER 865,063 949 911,552 pNOV4057 apoplast806,362 806 1,000,450

EXAMPLE 5

Phytase Activity in Seed from Maize Inbreds

Flour samples (1 gram) from several lines containing lead events wereextracted in 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM for 1 hour atambient temperature with stirring. Extraction volume was 100 ml.Extracts were clarified by centrifugation and diluted with sodiumacetate buffer (pH 5.5) Phytase activity was measured at pH 5.5 and 37 Cusing the method of Engelen et al. (2001) with modifications. Assayswere performed in microplates at a final reaction volume of 1 ml. Theresults are set forth in FIG. 7.

EXAMPLE 6

Feeding Trials

A formulation of phytase from corn seed is as corn flour or grist. FIGS.8A, 8B, and 8C summarize results from three feeding trials in whichchicken feed was supplemented with Nov9X phytase formulated as cornflour. Samples of transgenic corn seed containing extractable phytaseactivity were milled using a hammer or disc mill and were then addeddirectly to feed samples and mixed thoroughly. Treated feed was eitherused directly as mash feed (trial I) or was steam conditioned andpelleted (trials II and III). In all cases phytase corn flour was addedto feed rations that contained reduced levels of phosphate (negativecontrol). This allowed comparison of low phosphate diet (negativecontrol), high phosphate diet (positive control), and low phosphatediets supplemented with phytase formulated as milled corn.

The results in FIGS. 8 (A,B, and C) are plotted as feed conversionratios (FCRs). FCR refers to the amount of feed consumed divided by thenet weight gain of the chicken. A lower ratio indicates that a chickengained more weight per unit of feed consumed. A lower ratio indicatesthat a chicken more efficiently utilized the feed that was consumed.Standard poultry diets are used and two inorganic phosphate levels areincorporated into the diets, 0.450% (positive control) and 0.225%(negative control and enzyme-supplemented diets). The 0.45% level iscommonly used in commercial poultry diets. Replicate pens of 8 chickensfor each diet are grown until 21 days of age, and final weightsdetermined by subtracting the weight of the one day old chicks. Recordsare kept of the amount of feed consumed by each pen of chickens, and anaverage feed consumption is determined.

FIG. 8A shows results from a trial in which chickens were fed mashdiets. For this trial, Nov9X phytase was formulated by grinding wholetransgenic corn kernels to flour. Transgenic kernels were harvested fromplants containing vectors pNOV4057 (apoplast-targeted phytase) orpNOV4061 (ER-retained phytase)(see Table 2). Supplementation of lowphosphate diets with Nov9X phytase as corn flour improved FCR andrestored performance to levels equal to or better than that of thepositive control. These results demonstrate that Nov9X phytaseformulated as corn flour improves FCR in chickens fed a low phosphatediet.

FIG. 8B shows results from a trial in which chickens were fed pelletedfeed. For this trial Nov9x phytase formulated as corn flour was added tolow phosphate chicken feed before steam conditioning and pelleting. Asin FIG. 8A, the apoplast-targeted and ER-retained form of Nov9x phytasewere tested. Phytase supplementation restored performance to levelsequal to or better than that observed for the positive control. Theseresults demonstrate that Nov9x phytase synthesized directly in corn seedand formulated as corn flour improves FCR in chickens fed a lowphosphate diet.

FIG. 8C shows results from a trial in which the apoplast-targeted formof Nov9x phytase (encoded by vector pNOV4057) was formulated as a finegrind corn flour, a medium grind, or a coarse grind corn flour. Coarsegrind material consisted predominantly of particles >2000 microns;medium grind material was predominantly in the size range of 500-2000microns; and fine grind flour was <500 microns. The three formulationswere added to low phosphate rations prior to steam conditioning andpelleting as in FIG. 8B. Feed conversion ratios were improved at alldoses tested for all three formulations. And all three formulationsoutperformed the positive control at all doses tested. These resultsdemonstrate efficacy in chickens of the preferred formulation of Nov9Xphytase as cracked corn seed.

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1. A method of reducing feed conversion ratios or increasing weight gainof animals fed diets with inorganic phosphate at levels below 0.45%comprising: feeding to an animal a feed comprising inorganic phosphatebelow 0.45% and a phytase preparation prepared by expressing in a plantcell an expression cassette comprising a promoter operably linked to anucleic acid molecule encoding a polypeptide depicted in amino acids20-431 of SEQ ID NO. 4 or a conservative variant thereof.
 2. The methodof claim 1 wherein the phytase preparation is a liquid mixture.
 3. Themethod of claim 1 wherein the phytase preparation is a solid mixture. 4.The method of claim 1 wherein the preparation is transgenic plantmaterial.
 5. The method of claim 4 wherein the transgenic material istransgenic corn, grain, cracked corn, corn flour, or an enzyme extractprepared from corn.
 6. The method of claim 1 wherein the phytasepreparation further comprises at least one vitamin, mineral, an enzymeother than a thermotolerant phytase, an organic acid, probiotic(bacterial) or essential oil or a co-product.
 7. The method of claim 1wherein the phytase preparation is less than about 1% inorganicphosphate.